Recombinant RSV virus expression systems and vaccines

ABSTRACT

The present invention relates to genetically engineered recombinant RS viruses and viral vectors which contain heterologous genes which for the use as vaccines. In accordance with the present invention, the recombinant RS viral vectors and viruses are engineered to contain heterologous genes, including genes of other viruses, pathogens, cellular genes, tumor antigens, or to encode combinations of genes from different strains of RSV.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/724,388 filed Nov. 28, 2000, currently pending, which is acontinuation application of U.S. application Ser. No. 09/161,122 filedSep. 25, 1998, abandoned, which is a continuation-in-part application ofU.S. application Ser. No. 08/316,439 filed Sep. 30, 1994, now U.S. Pat.No. 5,840,520, issued Nov. 24, 1998. U.S. application Ser. No.09/161,122 filed Sep. 25, 1998 also claims benefit of priority from U.S.Provisional Application Ser. Nos. 60/060,153 filed Sep. 26, 1997;60/084,133 filed May 1, 1998; and 60/089,207 filed Jun. 12, 1998.

1. INTRODUCTION

The present invention relates to recombinant negative strand virus RNAtemplates which may be used to express heterologous gene products inappropriate host cell systems and/or to construct recombinant virusesthat express, package, and/or present the heterologous gene product. Theexpression products and chimeric viruses may advantageously be used invaccine formulations. In particular, the present invention relates tomethods of generating recombinant respiratory syncytial viruses and theuse of these recombinant viruses as expression vectors and vaccines. Theinvention is described by way of examples in which recombinantrespiratory syncytial viral genomes are used to generate infectiousviral particles.

2. BACKGROUND OF THE INVENTION

A number of DNA viruses have been genetically engineered to direct theexpression of heterologous proteins in host cell systems (e.g., vacciniavirus, baculovirus, etc.). Recently, similar advances have been madewith positive-strand RNA viruses (e.g., poliovirus). The expressionproducts of these constructs, i.e., the heterologous gene product or thechimeric virus which expresses the heterologous gene product, arethought to be potentially useful in vaccine formulations (either subunitor whole virus vaccines). One drawback to the use of viruses such asvaccinia for constructing recombinant or chimeric viruses for use invaccines is the lack of variation in its major epitopes. This lack ofvariability in the viral strains places strict limitations on therepeated use of chimeric vaccinia, in that multiple vaccinations willgenerate host-resistance to the strain so that the inoculated viruscannot infect the host. Inoculation of a resistant individual withchimeric vaccinia will, therefore, not induce immune stimulation.

By contrast, negative-strand RNA viruses such as influenza virus andrespiratory syncytial virus, demonstrate a wide variability of theirmajor epitopes. Indeed, thousands of variants of influenza have beenidentified; each strain evolving by antigenic drift. The negative-strandviruses such as influenza and respiratory syncytial virus would beattractive candidates for constructing chimeric viruses for use invaccines because its genetic variability allows for the construction ofa vast repertoire of vaccine formulations which will stimulate immunitywithout risk of developing a tolerance.

2.1 Respiratory Syncytial Virus

Virus families containing enveloped single-stranded RNA of thenegative-sense genome are classified into groups having non-segmentedgenomes (Paramyxoviridae, Rhabdoviridae) or those having segmentedgenomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae).Paramyxoviridae have been classified into three genera: paramyxovirus(sendai virus, parainfluenza viruses types 1-4, mumps, newcastle diseasevirus); morbillivirus (measles virus, canine distemper virus andrinderpest virus); and pneumovirus (respiratory syncytial virus andbovine respiratory syncytial virus).

Human respiratory syncytial virus (RSV) is the leading cause of severelower respiratory tract disease in infants and young children and isresponsible for considerable morbidity and mortality. Two antigenicallydiverse RSV subgroups A and B are present in human populations. RSV isalso recognized as an important agent of disease in immuno-compromisedadults and in the elderly. Due to the incomplete resistance to RSVreinfection induced by natural infection, RSV may infect multiple timesduring childhood and life. The goal of RSV immunoprophylaxis is toinduce sufficient resistance to prevent the serious disease which may beassociated with RSV infection. The current strategies for developing RSVvaccines principally revolve around the administration of purified viralantigen or the development of live attenuated RSV for intranasaladministration. However, to date there have been no approved vaccines orhighly effective antiviral therapy for RSV.

Infection with RSV can range from an unnoticeable infection to severepneumonia and death. RSV possesses a single-stranded nonsegmentednegative-sense RNA genome of 15,221 nucleotides (Collins, 1991, In Theparamyxoviruses pp. 103-162, D. W. Kingsbury (ed.) Plenum Press, NewYork). The genome of RSV encodes 10 mRNAs (Collins et al., 1984, J.Virol. 49: 572-578). The genome contains a 44 nucleotide leader sequenceat the 3′ termini followed by the NS1-NS2-N—P-M-SH-G-F-M2-L and a 155nucleotide trailer sequence at the 5′ termini (Collins. 1991, supra).Each gene transcription unit contains a short stretch of conserved genestart (GS) sequence and a gene end (GE) sequences.

The viral genomic RNA is not infectious as naked RNA. The RNA genome ofRSV is tightly encapsidated with the major nucleocapsid (N) protein andis associated with the phosphoprotein (P) and the large (L) polymerasesubunit. These proteins form the nucleoprotein core, which is recognizedas the minimum unit of infectivity (Brown et al., 1967, J. Virol. 1:368-373). The RSV N, P, and L proteins form the viral RNA dependent RNAtranscriptase for transcription and replication of the RSV genome (Yu etal., 1995, J. Virol. 69:2412-2419; Grosfeld et al., 1995, J. Virol.69:5677-86). Recent studies indicate that the M2 gene products (M2-1 andM2-2) are involved and are required for transcription (Collins et al.,1996, Proc. Natl. Acad. Sci. 93:81-5).

The M protein is expressed as a peripheral membrane protein, whereas theF and G proteins are expressed as integral membrane proteins and areinvolved in virus attachment and viral entry into cells. The G and Fproteins are the major antigens that elicit neutralizing antibodies invivo (as reviewed in McIntosh and Chanock, 1990 “Respiratory SyncytialVirus” 2nd ed. Virology (D. M. Knipe et al., Ed.) Raven Press, Ltd.,N.Y.). Antigenic dimorphism between the subgroups of RSV A and B ismainly linked to the G glycoprotein, whereas the F glycoprotein is moreclosely related between the subgroups.

Despite decades of research, no safe and effective RSV vaccine has beendeveloped for the prevention of severe morbidity and mortalityassociated with RSV infection. A formalin-inactivated virus vaccine hasfailed to provide protection against RSV infection and its exacerbatedsymptoms during subsequent infection by the wild-type virus in infants(Kapikian et al., 1969, Am. J. Epidemiol. 89:405-21; Chin et al., 1969,Am. J. Epidemiol. 89:449-63) Efforts since have focused on developinglive attenuated temperature-sensitive mutants by chemical mutagenesis orcold passage of the wild-type RSV (Gharpure et al., 1969, J. Virol. 3:414-21; Crowe et al., 1994, Vaccine 12: 691-9). However, earlier trialsyielded discouraging results with these live attenuated temperaturesensitive mutants. Virus candidates were either underattenuated oroverattenuated (Kim et al., 1973, Pediatrics 52:56-63; Wright et al.,1976, J. Pediatrics 88:931-6) and some of the vaccine candidates weregenetically unstable which resulted in the loss of the attenuatedphenotype (Hodes et al., 1974, Proc. Soc. Exp. Biol. Med. 145:1158-64).

Attempts have also been made to engineer recombinant vaccinia vectorswhich express RSV F or G envelope glycoproteins. However, the use ofthese vectors as vaccines to protect against RSV infection in animalstudies has shown inconsistent results (Olmsted et al. 1986, Proc. Natl.Acad. Sci. 83:7462-7466; Collins et al., 1990, Vaccine 8:164-168).

Thus, efforts have turned to engineering recombinant RSV to generatevaccines. For a long time, negative-sense RNA viruses were refractory tostudy. Only recently has it been possible to recover negative strand RNAviruses using a recombinant reverse genetics approach (U.S. Pat. No.5,166,057 to Palese et al.). Although this method was originally appliedto engineer influenza viral genomes (Luytjes et al. 1989, Cell59:1107-1113; Enami et al. 1990, Proc. Natl. Acad. Sci. USA 92:11563-11567), it has been successfully applied to a wide variety ofsegmented and nonsegmented negative strand RNA viruses, including rabies(Schnell et al. 1994, EMBO J. 13: 4195-4203); VSV (Lawson et al., 1995,Proc. Natl. Acad. Sci USA 92: 4477-81); measles virus (Radecke et al.,1995, EMBO J. 14:5773-84); rinderpest virus (Baron & Barrett, 1997, J.virol. 71: 1265-71); human parainfluenza virus (Hoffman & Banerjee,1997, J. Virol. 71:3272-7; Dubin et al., 1997, Virology 235:323-32); SV5(He et al., 1997, Virology 237:249-60); respiratory syncytial virus(Collins et al. 1991, Proc. Natl. Acad. Sci. USA 88: 9663-9667) andSendai virus (Park et al. 1991, Proc. Natl. Acad. Sci. USA 88:5537-5541;Kato et al. 1996, Genes to Cells 1:569-579). Although this approach hasbeen used to successfully rescue RSV, a number of groups have reportedthat RSV is still refractory to study given several properties of RSVwhich distinguish it from the better characterized paramyxoviruses ofthe genera Paramyxovirus, Rubulavirus, and Morbillivirus. Thesedifferences include a greater number of RNAs, an unusual gene order atthe 3′ end of the genome, extensive strain-to-strain sequence diversity,several proteins not found in other nonsegmented negative strand RNAviruses and a requirement for the M2 protein (ORF1) to proceed with fullprocessing of full length transcripts and rescue of a full length genome(Collins et al. PCT WO97/12032; Collins, P. L. et al. pp 1313-1357 ofvolume 1, Fields Virology, et al., Eds. (3rd ed., Raven Press, 1996).

3. SUMMARY OF THE INVENTION

The present invention relates to genetically engineered recombinant RSviruses and viral vectors which contain heterologous genes which for theuse as vaccines. In accordance with the present invention, therecombinant RS viral vectors and viruses are engineered to containheterologous genes, including genes of other viruses, pathogens,cellular genes, tumor antigens, or to encode combinations of genes fromdifferent strains of RSV.

Recombinant negative-strand viral RNA templates are described which maybe used to transfect transformed cell that express the RNA dependent RNApolymerase and allow for complementation. Alternatively, a plasmidexpressing the components of the RNA polymerase from an appropriatepromoter can be used to transfect cells to allow for complementation ofthe negative-strand viral RNA templates. Complementation may also beachieved with the use of a helper virus or wild-type virus to providethe RNA dependent RNA polymerase. The RNA templates are prepared bytranscription of appropriate DNA sequences with a DNA-directed RNApolymerase. The resulting RNA templates are of negative-orpositive-polarity and contain appropriate terminal sequences whichenable the viral RNA-synthesizing apparatus to recognize the template.Bicistronic mRNAs can be constructed to permit internal initiation oftranslation of viral sequences and allow for the expression of foreignprotein coding sequences from the regular terminal initiation site, orvice versa.

As demonstrated by the examples described herein, recombinant RSV genomein the positive-sense or negative-sense orientation is co-transfectedwith expression vectors encoding the viral nucleocapsid (N) protein, theassociated nucleocapsid phosphoprotein (P), the large (L) polymerasesubunit protein, with or without the M2/ORF1 protein of RSV to generateinfectious viral particles. Plasmids encoding RS virus polypeptides areused as the source of proteins which were able to replicate andtranscribe synthetically derived RNPs. The minimum subset of RSVproteins needed for specific replication and expression of the viral RNPwas found to be the three polymerase complex proteins (N, P and L). Thissuggests that the entire M2 gene function may not be absolutely requiredfor the replication, expression and rescue of infectious RSV.

The expression products and/or chimeric virions obtained mayadvantageously be utilized in vaccine formulations. In particular,recombinant RSV genetically engineered to demonstrate an attenuatedphenotype may be utilized as a live RSV vaccine. In another embodimentof the invention, recombinant RSV may be engineered to express theantigenic polypeptides of another strain of RSV (e.g., RSV G and Fproteins) or another virus (e.g., an immunogenic peptide from gp120 ofHIV) to generate a chimeric RSV to serve as a vaccine, that is able toelicit both vertebrate humoral and cell-mediated immune responses. Theuse of recombinant influenza or recombinant RSV for this purpose isespecially attractive since these viruses demonstrate tremendous strainvariability allowing for the construction of a vast repertoire ofvaccine formulations. The ability to select from thousands of virusvariants for constructing chimeric viruses obviates the problem of hostresistance encountered when using other viruses such as vaccinia.

The invention provides a genetically manipulated, replicating,infectious paramxyovirus, comprising at least one alteration to theviral genome, selected from an insertion and a deletion. In certainembodiments the viral genome is derived from respiratory syncytialvirus. In certain embodiments, the viral genome is derived fromparainfluenza virus.

The invention provides a process for the preparation of an infectiousreplicating paramxyovirus comprising the steps of: providing a host cellexpressing a heterologous RNA polymerase; introducing into said hostcell: 1. one or more DNA molecules encoding the virus N, P and Lproteins operably linked to a polymerase binding site; 2. a DNA moleculecomprising the cDNA of the paramxyovirus wherein the cDNA encodes theentire genome of the virus or is modified by the incorporation of amutation or heterologous sequence, and wherein the DNA molecule istranscribed by a heterologous RNA polymerase, and isolating the virusproduced by the cell. In certain embodiments, the virus is respiratorysyncytial virus. In certain embodiments the virus is parainfluenzavirus.

3.1 Definitions

As used herein, the following terms will have the meanings indicated:

cRNA=anti-genomic RNA

HA=hemagglutinin (envelope glycoprotein)

HIV=human immunodefiency virus

L=large polymerase subunit

M=matrix protein (lines inside of envelope)

MDCK=Madin Darby canine kidney cells

MDBK=Madin Darby bovine kidney cells

moi=multiplicity of infection

N=nucleocapsid protein

NA=neuraminidase (envelope glycoprotein)

NP=nucleoprotein (associated with RNA and required for polymeraseactivity)

NS=nonstructural protein (function unknown)

nt=nucleotide

P=nucleocapsid phosphoprotein

PA, PB1, PB2=RNA-directed RNA polymerase components

RNP=ribonucleoprotein (RNA, PB2, PB 1, PA and NP)

rRNP=recombinant RNP

RSV=respiratory syncytial virus

vRNA=genomic virus RNA

viral polymerase complex=PA, PB1, PB2 and NP

WSN=influenza A/WSN/33 virus

WSN-HK virus: reassortment virus containing seven genes from WSN virusand the NA gene from influenza A/HK/8/68 virus

4. DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the RSV/CAT construct (pRSVA2CAT)used in rescue experiments. The approximate 100 nt long leader and 200nt long trailer regions of RSV were constructed by the controlledannealing of synthetic oligonucleotides containing partial overlappingcomplementarity. The overlapping leader oligonucleotides are indicatedby the 1L -5L (SEQ ID NOS:1-5) shown in the construct. The overlappingtrailer nucleotides are indicated by the 1T -9T (SEQ ID NOS:6-14) shownin the construct. The nucleotide sequences of the leader and trailerDNAs were ligated into purified CAT gene DNA at the indicate XbaI andPstI sites respectively. This entire construct was then ligated intoKpnI/HindIII digested pUC19. The inclusion of a T7 promoter sequence anda HgaI site flanking the trailer and leader sequences, respectively,allowed in vitro synthesis of RSV/CAT RNA transcripts containing theprecise genomic sequence 3′ and 5′ ends.

FIG. 2. Thin layer chromatogram (TLC) showing the CAT activity presentin 293 cell extracts following infection and transfection with RNAtranscribed from the RSV/CAT construct shown in FIG. 11. Confluentmonolayers of 293 cells in six-well plates (−10⁶ cells) were infectedwith either RSV A2 or B9320 at an m.o.i. of 0.1-1.0 pfu cell. At 1 hourpost infection cells were transfected with 5-10 μg of CAT/RSV using theTransfect-Act™ protocol of Life Technologies. At 24 hours post infectionthe infected/transfected monolayers were harvested and processed forsubsequence CAT assay according to Current Protocols in MolecularBiology, Vol. 1, Chapter 9.6.2; Gorman, et al., (1982) Mol. Cell. Biol.2:1044-1051. Lanes 1, 2, 3 and 4 show the CAT activity present in (1)uninfected 293 cells, transfected with CAT/RSV-A2 infected 293 cells,co-infected with supernatant from (2) above. The CAT activity observedin each lane was produced from ⅕ of the total cellular extract from 10⁶cells.

FIG. 3. Schematic representation of the RSV strain A2 genome showing therelative positions of the primer pairs used for the synthesis of cDNAscomprising the entire genome (SEQ ID NOS:5-28). The endonuclease sitesused to splice these clones together are indicated; these sites werepresent in the native RSV sequence and were included in the primers usedfor cDNA synthesis. Approximately 100 ng of viral genomic RNA was usedin RT/PCR reactions for the separate synthesis of each of the sevencDNAs. The primers for the first and second strand cDNA synthesis fromthe genomic RNA template are also shown. For each cDNA, the primers forthe first strand synthesis are nos. 1-7 and the primers for the secondstrand synthesis are nos. 1′-7′.

FIG. 4. Schematic representation of the RSV subgroup B strain B9320.BamH1 sites were created in the oligonucleotide primers used for RT/PCRin order to clone the G and F genes from the B9320 strain into RSVsubgroup A2 antigenomic cDNA (FIG. 4A). A cDNA fragment which containedG and F genes from 4326 nucleotides to 9387 nucleotides of A2 strain wasfirst subcloned into pUC19 (pUCRVH). Bgl II sites were created atpositions of 4630 (SH/G intergenic junction) (FIG. 4B) and 7554 (F/M2intergenic junction (FIG. 4C). B93260 A-G and -F cDNA inserted intopUCR/H which is deleted of the A-G and F genes. The resultingantigenomic cDNA clone was termed as pRSVB-GF and was used to transfectHep-2 cells to generate infectious RSVB-GF virus.

FIG. 5. Recombinant RSVB-GF virus was characterized by RT/PCR using RSVsubgroup B specific primers. RSV subgroup B specific primers in the Gregion were incubated with aliquots of the recombinant RSV viral genomesand subjected to PCR. The PCR products were analyzed by electrophoresison a 1% agarose gel and visualized by staining with ethidium bromide. Asshown, no DNA product was produced in the RT/PCR reaction using RSV A2as a template. However, a predicted product of 254 base pairs was seenin RT/PCR of RSVB-GF RNA and PCR control of plasmid pRSV-GF DNA astemplate, indicating the rescued virus contained G and F genes derivedfrom B9320 virus.

FIG. 6. Identification of chimeric (rRSVA2(B-G) by RT/PCR and Northernblot analysis of RNA expression. FIG. 6A. RT/PCT analysis of chimericrRSV A2(B-G), A2(B-G), in comparison with wild-type A2(A2). Virion RNAextracted from rRSVA2(B-G) (lanes 1, 2) and rRSVA2 (lanes 3,4) wasreverse transcribed using a primer annealed to (−) sense vRNA in the RSVF gene in the presence (+) or absence (−) of reverse transcriptase (RT),followed by PCR with a primer fair flanking the B-G insertion site. NoDNA was detected in RT/PCR when reverse transcriptase (RT) was absent(lanes 2,4). A cDNA fragment, which is about 1 kb bigger than the cDNAderived from A2, was produced from rRSVA(B-G). This longer PCR DNAproduct was digested by Stu I restriction enzyme unique to the insertedB-G gene (lane 5). 100 bp DNA size marker is indicated (M). FIG. 6B.Northern blot analysis of G mRNA expression. Hep-2 cells were infectedwith RSV B9320, rRSV and chimeric rRSV A2 (B-G). At 48 hr postinfection,total cellular RNA was extracted and electrophoresed on a 1.2% agarosegel containing formaldehyde. RNA was transferred to Hybond Nylonmembrane and the filter was hybridized with a ³²P-labeledoligonucleotide probe specific for A2-G or specific for B9320-G mRNA.Both A2 G specific and B9320 G specific transcripts were detected in therRSV A2 (B-G) infected cells. The run-off RNA transcript (G-M2) fromrRSV A2 (B-G) infected cells is also indicated.

FIG. 7. Analysis of protein expression by rRSV A2 (B-G). Hep-2 cellswere mock-infected (lanes 1, 5), infected with RSV B9320 (lanes 2, 6),rRSV (lanes 3, 7) and rRSV A2 (B-G) (lanes 4, 8). At 14-18 hrpostinfection, infected cells were labeled with ³⁵S-promix andpolypeptides were immunoprecipitated by goat polyclonal antiserumagainst RSV A2 strain (lanes 1-5) or by mouse polyclonal antiserumagainst RSV B9320 strain (lanes 5-8). Immunoprecipitated polypeptideswere separated on a 10% polyacrylamide gel. Both RSV A2 specific Gprotein and RSV B9320 specific G protein were produced in rRSV A2 (B-G)infected cells. The G protein migration is indicated by *. Mobility ofthe F1 glycoprotein, and N, P, and M is indicated. Molecular sizes areshown on the left in kilodaltons.

FIG. 8. Plaque morphology of rRSV, rRSVC3G, rRSV A2 (B-G) and wild-typeA2 virus (wt A2). Hep-2 cells were infected with each virus andincubated at 35° C. for six days. The cell monolayers were fixed,visualized by immunostaining, and photographed.

FIG. 9. Growth curve of rRSV, rRSVC4G, wild-type A2 RSV (wt A2) andchimeric rRSV A2 (B-G). Hep-2 cells were infected with either virus at amoi of 0.5 and the medium was harvested at 24 hr intervals. The titer ofeach virus was determined in duplicate by plaque assay on Hep-2 cellsand visualized by immunostaining.

FIG. 10. RSV L (SEQ ID NO:29) protein charged residue clusters targetedfor site-directed mutagenesis. Contiguous charged amino acid residues inclusters were converted to alanines by site-directed mutagenesis of theRSV L gene using the QuikChange site-directed mutagenesis kit(Stratagene).

FIG. 11. RSV L (SEQ ID NO:29) protein cysteine residues targeted forsite-directed mutagenesis. Cysteine residues were converted toalanine-residues by site-directed mutagenesis of the RSV L gene usingthe QuikChange site-directed mutagenesis kit (Stratagene).

FIG. 12. Identification RSV M2-2 and SH deletion mutants. Deletions inM2-2 were generated by Hind III digestion of pET(S/B) followed byrecloning of a remaining Sac I to BamHI fragment into a full-lengthclone. Deletions in SH were generated by Sac I digestion of pET(A/S)followed by recloning of a remaining Avr II Sac I fragment into afull-length clone. FIG. 12A. Identification of the recovered rRSVsSH andrRSV M2-2 was performed by RT/PCR using primer pairs specific for the SHgene or M2-2 gene, respectively. FIG. 12B rRSV SH M2-2 was also detectedby RT/PCR using primer pairs specific for the M2-2 and SH genes. RT/PCRproducts were run on an ethidium bromide agarose gel and bands werevisualized by ultraviolet (UV) light.

5. DESCRIPTION OF THE INVENTION

The present invention relates to genetically engineered recombinant RSviruses and viral vectors which express heterologous genes or mutated RSviral genes or a combination of viral genes derived from differentstrains of RS virus. The invention relates to the construction and useof recombinant negative strand RS viral RNA templates which may be usedwith viral RNA-directed RNA polymerase to express heterologous geneproducts in appropriate host cells and/or to rescue the heterologousgene in virus particles. The RNA templates of the present invention maybe prepared by transcription of appropriate DNA sequences using aDNA-directed RNA polymerase such as bacteriophage T7, T3 or Sp6polymerase. The recombinant RNA templates may be used to transfectcontinuous/transfected cell lines that express the RNA-directed RNApolymerase proteins allowing for complementation.

The invention is demonstrated by way of working examples in whichinfectious RSV is rescued from cDNA containing the RSV genome in thegenomic or antigenomic sense introduced into cells expressing the N, P,and L proteins of the RSV polymerase complex. The working examplesfurther demonstrate that expression of M2-2 is not required for recoveryof infectious RSV from cDNA which is contrary to what has been reportedearlier (Collins et al., 1995, Proc. Natl. Acad. Sci. USA 92:11563-7).Furthermore, the deletion of the M2-ORF2 minus recombinant RSV cDNAresults in the rescue of attenuated RSV particles. M2-deleted-RSV is anexcellent vehicle to generate chimeric RSV encoding heterologous geneproducts in place of the M2 genes, these chimeric viral vectors andrescued virus particles have utility as expression vectors for theexpression of heterologous gene products and as live attenuated RSVvaccines expressing either RSV antigenic polypeptides or antigenicpolypeptides of other viruses.

The invention is further demonstrated by way of working examples inwhich a cDNA clone which contained the complete genome of RSV, inaddition to a T7 promoter, a hepatitis delta virus ribozyme and a T7terminator is used to generate an infectious viral particle whenco-transfected with expression vectors encoding the N, P, L and/orM2-ORF1 proteins of RSV. In addition, the working examples describe RNAtranscripts of cloned DNA containing the coding region—in negative senseorientation—of the chloramphenicol-acetyl-transferase (CAT) gene or thegreen fluorescent protein (GFP) gene flanked by the 5′ terminal and 3′terminal nucleotides of the RSV genome. The working examples furtherdemonstrate that an RSV promoter mutated to have increased activityresulted in rescue of infectious RSV particles from a full length RSVcDNA with high efficiency. These results demonstrate the successful useof recombinant viral negative strand templates and RSV polymerase withincreased activity to rescue RSV. This system is an excellent tool toengineer RSV viruses with defined biological properties, e.g.live-attenuated vaccines against RSV, and to use recombinant RSV as anexpression vector for the expression of heterologous gene products.

This invention relates to the construction and use of recombinantnegative strand viral RNA templates which may be used with viralRNA-directed RNA polymerase to express heterologous gene products inappropriate host cells, to rescue the heterologous gene in virusparticles and/or express mutated or chimeric recombinant negative strandviral RNA templates (see U.S. Pat. No. 5,166,057 to Palese et al.,incorporated herein by reference in its entirety). In a specificembodiment of the invention, the heterologous gene product is a peptideor protein derived from another strain of the virus or another virus.The RNA templates may be in the positive or negative-sense orientationand are prepared by transcription of appropriate DNA sequences using aDNA-directed RNA polymerase such as bacteriophage T7, T3 or the Sp6polymerase.

The ability to reconstitute RNP's in vitro allows the design of novelchimeric influenza and RSV viruses which express foreign genes. One wayto achieve this goal involves modifying existing viral genes. Forexample, the G or F gene may be modified to contain foreign sequences,such as the HA gene of influenza in its external domains. Where theheterologous sequence are epitopes or antigens of pathogens, thesechimeric viruses may be used to induce a protective immune responseagainst the disease agent from which these determinants are derived. Forexample, a chimeric RNA may be constructed in which a coding sequencederived from the gp120 coding region of human immunodeficiency virus wasinserted into the coding sequence of RSV, and chimeric virus producedfrom transfection of this chimeric RNA segment into a host cell infectedwith wild-type RSV.

In addition to modifying genes coding for surface proteins, genes codingfor nonsurface proteins may be altered. The latter genes have been shownto be associated with most of the important cellular immune responses inthe RS virus system. Thus, the inclusion of a foreign determinant in theG or F gene of RSV may—following infection—induce an effective cellularimmune response against this determinant. Such an approach may beparticularly helpful in situations in which protective immunity heavilydepends on the induction of cellular immune responses (e.g., malaria,etc.).

The present invention also relates to attenuated recombinant RSVproduced by introducing specific mutations in the genome of RSV whichresults in an amino acid change in an RSV protein, such as a polymeraseprotein, which results in an attenuated phenotype.

5.1 Construction of the Recombinant RNA Templates

Heterologous gene coding sequences flanked by the complement of theviral polymerase binding site/promoter, e.g., the complement of the3′-RSV termini or the 3′- and 5′-RSV termini may be constructed usingtechniques known in the art. Heterologous gene coding sequences may alsobe flanked by the complement of the RSV polymerase bindingsite/promoter, e.g., the leader and trailer sequence of RSV usingtechniques known in the art. Recombinant DNA molecules containing thesehybrid sequences can be cloned and transcribed by a DNA-directed RNApolymerase, such as bacteriophage T7, T3 or the Sp6 polymerase and thelike, to produce the recombinant RNA templates which possess theappropriate viral sequences that allow for viral polymerase recognitionand activity.

In a preferred embodiment of the present invention, the heterologoussequences are derived from the genome of another strain of RSV, e.g.,the genome of RSV A strain is engineered to include the nucleotidesequences encoding the antigenic polypeptides G and F of RSV B strain,or fragments thereof. In such an embodiment of the invention,heterologous coding sequences from another strain of RSV can be used tosubstitute for nucleotide sequences encoding antigenic polypeptides ofthe starting strain, or be expressed in addition to the antigenicpolypeptides of the parent strain, so that a recombinant RSV genome isengineered to express the antigenic polypeptides of one, two or morestrains of RSV.

In yet another embodiment of the invention, the heterologous sequencesare derived from the genome of any strain of influenza virus. Inaccordance with the present invention, the heterologous coding sequencesof influenza may be inserted within a RSV coding sequence such that achimeric gene product is expressed which contains the heterologouspeptide sequence within the RSV viral protein. In either embodiment, theheterologous sequences derived from the genome of influenza may include,but are not limited to HA, NA, PB1, PB2, PA, NS1 or NS2.

In one specific embodiment of the invention, the heterologous sequencesare derived from the genome of human immunodeficiency virus (HIV),preferably human immunodeficiency virus-1 or human immunodeficiencyvirus-2. In another embodiment of the invention, the heterologous codingsequences may be inserted within an RSV gene coding sequence such that achimeric gene product is expressed which contains the heterologouspeptide sequence within the influenza viral protein. In such anembodiment of the invention, the heterologous sequences may also bederived from the genome of a human immunodeficiency virus, preferably ofhuman immunodeficiency virus-1 or human immunodeficiency virus-2.

In instances whereby the heterologous sequences are HIV-derived, suchsequences may include, but are not limited to sequences derived from theenv gene (i.e., sequences encoding all or part of gp160, gp120, and/orgp41), the pol gene (i.e., sequences encoding all or part of reversetranscriptase, endonuclease, protease, and/or integrase), the gag gene(i.e., sequences encoding all or part of p7, p6, p55, p17/18, p24/25)tat, rev, nef, vif, vpu, vpr, and/or vpx.

One approach for constructing these hybrid molecules is to insert theheterologous coding sequence into a DNA complement of a RSV genomic RNAso that the heterologous sequence is flanked by the viral sequencesrequired for viral polymerase activity; i.e., the viral polymerasebinding site/promoter, hereinafter referred to as the viral polymerasebinding site. In an alternative approach, oligonucleotides encoding theviral polymerase binding site, e.g., the complement of the 3′-terminusor both termini of the virus genomic segments can be ligated to theheterologous coding sequence to construct the hybrid molecule. Theplacement of a foreign gene or segment of a foreign gene within a targetsequence was formerly dictated by the presence of appropriaterestriction enzyme sites within the target sequence. However, recentadvances in molecular biology have lessened this problem greatly.Restriction enzyme sites can readily be placed anywhere within a targetsequence through the use of site-directed mutagenesis (e.g., see, forexample, the techniques described by Kunkel, 1985, Proc. Natl. Acad.Sci. U.S.A. 82;488). Variations in polymerase chain reaction (PCR)technology, described infra, also allow for the specific insertion ofsequences (i.e., restriction enzyme sites) and allow for the facileconstruction of hybrid molecules. Alternatively, PCR reactions could beused to prepare recombinant templates without the need of cloning. Forexample, PCR reactions could be used to prepare double-stranded DNAmolecules containing a DNA-directed RNA polymerase promoter (e.g.,bacteriophage T3, T7 or Sp6) and the hybrid sequence containing theheterologous gene and the influenza viral polymerase binding site. RNAtemplates could then be transcribed directly from this recombinant DNA.In yet another embodiment, the recombinant RNA templates may be preparedby ligating RNAs specifying the negative polarity of the heterologousgene and the viral polymerase binding site using an RNA ligase. Sequencerequirements for viral polymerase activity and constructs which may beused in accordance with the invention are described in the subsectionsbelow.

5.1.1 Insertion of the Heterologous Genes

The gene coding for the L protein contains a single open reading frame.The genes coding for NS or M2 contain two open reading frames for NS1and NS2 and ORF1 and 2, respectively. The G and F proteins, coded for byseparate genes, are the major surface glycoproteins of the virus.Consequently, these proteins are the major targets for the humoralimmune response after infection. Insertion of a foreign gene sequenceinto any of these coding regions could be accomplished by either anaddition of the foreign sequences to be expressed or by a completereplacement of the viral coding region with the foreign gene or by apartial replacement. The heterologous sequences inserted into the RSVgenome may be any length up to approximately 5 kilobases. Completereplacement would probably best be accomplished through the use ofPCR-directed mutagenesis.

Alternatively, a bicistronic mRNA could be constructed to permitinternal initiation of translation of viral sequences and allow for theexpression of foreign protein coding sequences from the regular terminalinitiation site. Alternatively, a bicistronic mRNA sequence may beconstructed wherein the viral sequence is translated from the regularterminal open reading frame, while the foreign sequence is initiatedfrom an internal site. Certain internal ribosome entry site (IRES)sequences may be utilized. The IRES sequences which are chosen should beshort enough to not interfere with RS virus packaging limitations. Thus,it is preferable that the IRES chosen for such a bicistronic approach beno more than 500 nucleotides in length, with less than 250 nucleotidesbeing preferred. Further, it is preferable that the IRES utilized notshare sequence or structural homology with picornaviral elements.Preferred IRES elements include, but are not limited to the mammalianBiP IRES and the hepatitis C virus IRES.

5.2 Expression of Heterologous Gene Products using Recombinant RNATemplate

The recombinant templates prepared as described above can be used in avariety of ways to express the heterologous gene products in appropriatehost cells or to create chimeric viruses that express the heterologousgene products. In one embodiment, the recombinant template can becombined with viral polymerase complex purified infra, to produce rRNPswhich are infectious. To this end, the recombinant template can betranscribed in the presence of the viral polymerase complex.Alternatively, the recombinant template may be mixed with or transcribedin the presence of viral polymerase complex prepared using recombinantDNA methods (e.g. see Kingsbury et al., 1987, Virology 156:396-403). Inyet another embodiment, the recombinant template can be used totransfect appropriate host cells to direct the expression of theheterologous gene product at high levels. Host cell systems whichprovide for high levels of expression include continuous cell lines thatsupply viral functions such as cell lines superinfected with RSV, celllines engineered to complement RSV viral functions, etc.

5.3 Preparation of Chimeric Negative Strand RNA Virus

In order to prepare chimeric virus, reconstituted RNPs containingmodified RSV RNAs or RNA coding for foreign proteins may be used totransfect cells which are also infected with a “parent” RSV virus.Alternatively, the reconstituted RNP preparations may be mixed with theRNPs of wild type parent virus and used for transfection directly.Following reassortment, the novel viruses may be isolated and theirgenomes be identified through hybridization analysis. In additionalapproaches described herein for the production of infectious chimericvirus, rRNPs may be replicated in host cell systems that express the RSVor influenza viral polymerase proteins (e.g., in virus/host cellexpression systems; transformed cell lines engineered to express thepolymerase proteins, etc.), so that infectious chimeric virus arerescued; in this instance, helper virus need not be utilized since thisfunction is provided by the viral polymerase proteins expressed. In aparticularly desirable approach, cells infected with rRNPs engineeredfor all eight influenza virus segments may result in the production ofinfectious chimeric virus which contain the desired genotype; thuseliminating the need for a selection system.

Theoretically, one can replace any one of the genes of RSV, or part ofany one of the RSV genes, with the foreign sequence. However, anecessary part of this equation is the ability to propagate thedefective virus (defective because a normal viral gene product ismissing or altered). A number of possible approaches exist to circumventthis problem.

A third approach to propagating the recombinant virus may involveco-cultivation with wild-type virus. This could be done by simply takingrecombinant virus and co-infecting cells with this and another wild-typevirus (preferably a vaccine strain). The wild-type virus shouldcomplement for the defective virus gene product and allow growth of boththe wild-type and recombinant virus. This would be an analogoussituation to the propagation of defective-interfering particles ofinfluenza virus (Nayak et al., 1983, In: Genetics of Influenza Viruses,P. Palese and D. W. Kingsbury, eds., Springer-Verlag, Vienna, pp.255-279). In the case of defective-interfering viruses, conditions canbe modified such that the majority of the propagated virus is thedefective particle rather than the wild-type virus. Therefore thisapproach may be useful in generating high titer stocks of recombinantvirus. However, these stocks would necessarily contain some wild-typevirus.

Alternatively, synthetic RNPs may be replicated in cells co-infectedwith recombinant viruses that express the RS virus polymerase proteins.In fact, this method may be used to rescue recombinant infectious virusin accordance with the invention. To this end, the RSV virus polymeraseproteins may be expressed in any expression vector/host cell system,including but not limited to viral expression vectors (e.g., vacciniavirus, adenovirus, baculovirus, etc.) or cell lines that express thepolymerase proteins (e.g., see Krystal et al., 1986, Proc. Natl. Acad.Sci. USA 83: 2709-2713).

5.4 Generation of Chimeric Viruses with an Attenuated Phenotype

The methods of present invention may be used to introduce mutations orheterologous sequences to generate chimeric attenuated viruses whichhave many applications, including analysis of RSV molecular biology,pathogenesis, and growth and infection properties. In accordance withthe present invention, mutations or heterologous sequences may beintroduced for example into the F or G protein coding sequences, NS1,NS2, M1ORF1, M2ORF2, N, P, or L coding sequences. In yet anotherembodiment of the present invention, a particular viral gene, or theexpression thereof, may be eliminated to generate an attenuatedphenotype, e.g., the M ORF may be deleted from the RSV genome togenerate a recombinant RSV with an attenuated phenotype. In yet anotherembodiment, the individual internal genes of human RSV can be replacedby another strains counterpart, or their bovine or murine counterpart.This may include part or all of one or more of the NS1, NS2, N, P, M,SH, M2(ORF1), M2(ORF2) and L genes or the G and F genes. The RSV genomecontains ten mRNAs encoding three transmembrane proteins, G protein,fusion F protein required for penetration, and the small SH protein; thenucleocapsid proteins N, P and L; transcription elongation factor M2 ORF1; the matrix M protein and two nonstructural proteins, NS1 and NS2. Anyone of the proteins may be targeted to generate and attenuatedphenotype. Other mutations which may be utilized to result in anattenuated phenotype are insertional, deletional and site directedmutations of the leader and trailer sequences.

In accordance with the present invention, an attenuated RSV exhibits asubstantially lower degree of virulence as compared to a wild-typevirus, including a slower growth rate, such that the symptoms of viralinfection do not occur in an immunized individual.

In accordance with the present invention attenuated recombinant RSV maybe generated by incorporating a broad range of mutations includingsingle nucleotide changes, site-specific mutations, insertions,substitutions, deletions, or rearrangements. These mutations may affecta small segment of the RSV genome, e.g., 15 to 30 nucleotides, or largesegments of the RSV genome, e.g., 50 to 1000 nucleotides, depending onthe nature of the mutation. In yet another embodiment, mutations areintroduced upstream or downstream of an existing cis-acting regulatoryelement in order to ablate its activity, thus resulting in an attenuatedphenotype.

In accordance with the invention, a non-coding regulatory region of avirus can be altered to down-regulate any viral gene, e.g. reducetranscription of its mRNA and/or reduce replication of vRNA (viral RNA),so that an attenuated virus is produced.

Alterations of non-coding regulatory regions of the viral genome whichresult in down-regulation of replication of a viral gene, and/ordown-regulation of transcription of a viral gene will result in theproduction of defective particles in each round of replication; i.e.particles which package less than the full complement of viral segmentsrequired for a fully infectious, pathogenic virus. Therefore, thealtered virus will demonstrate attenuated characteristics in that thevirus will shed more defective particles than wild type particles ineach round of replication. However, since the amount of proteinsynthesized in each round is similar for both wild type virus and thedefective particles, such attenuated viruses are capable of inducing agood immune response.

The foregoing approach is equally applicable to both segmented andnon-segmented viruses, where the down regulation of transcription of aviral gene will reduce the production of its mRNA and the encoded geneproduct. Where the viral gene encodes a structural protein, e.g., acapsid, matrix, surface or envelope protein, the number of particlesproduced during replication will be reduced so that the altered virusdemonstrates attenuated characteristics; e.g., a titer which results insubclinical levels of infection. For example, a decrease in viral capsidexpression will reduce the number of nucleocapsids packaged duringreplication, whereas a decrease in expression of the envelope proteinmay reduce the number and/or infectivity of progeny virions.Alternatively, a decrease in expression of the viral enzymes requiredfor replication, e.g., the polymerase, replicase, helicase, and thelike, should decrease the number of progeny genomes generated duringreplication. Since the number of infectious particles produced duringreplication are reduced, the altered viruses demonstrated attenuatedcharacteristics. However, the number of antigenic virus particlesproduced will be sufficient to induce a vigorous immune response.

An alternative way to engineer attenuated viruses involves theintroduction of an alteration, including but not limited to aninsertion, deletion or substitution of one or more amino acid residuesand/or epitopes into one or more of the viral proteins. This may bereadily accomplished by engineering the appropriate alteration into thecorresponding viral gene sequence. Any change that alters the activityof the viral protein so that viral replication is modified or reducedmay be accomplished in accordance with the invention.

For example, alterations that interfere with but do not completelyabolish viral attachment to host cell receptors and ensuing infectioncan be engineered into viral surface antigens or viral proteasesinvolved in processing to produce an attenuated strain. According tothis embodiment, viral surface antigens can be modified to containinsertions, substitution or deletions of one or more amino acids orepitopes that interfere with or reduce the binding affinity of the viralantigen for the host cell receptors. This approach offers an addedadvantage in that a chimeric virus which expresses a foreign epitope maybe produced which also demonstrates attenuated characteristics. Suchviruses are ideal candidates for use as live recombinant vaccines. Forexample, heterologous gene sequences that can be engineered into thechimeric viruses of the invention include, but are not limited to,epitopes of human immunodeficiency virus (HIV) such as gp120; hepatitisB virus surface antigen (HBsAg); the glycoproteins of herpes virus(e.g., gD, gE); VP1 of poliovirus; and antigenic determinants ofnonviral pathogens such as bacteria and parasites, to name but a few.

In this regard, RSV is an ideal system in which to engineer foreignepitopes, because the ability to select from thousands of virus variantsfor constructing chimeric viruses obviates the problem of hostresistance or immune tolerance encountered when using other virusvectors such as vaccinia. In another embodiment, alterations of viralproteases required for processing viral proteins can be engineered toproduce attenuation. Alterations which affect enzyme activity and renderthe enzyme less efficient in processing, should affect viralinfectivity, packaging, and/or release to produce an attenuated virus.

In another embodiment, viral enzymes involved in viral replication andtranscription of viral genes, e.g., viral polymerases, replicases,helicases, etc. may be altered so that the enzyme is less efficient oractive. Reduction in such enzyme activity may result in the productionof fewer progeny genomes and/or viral transcripts so that fewerinfectious particles are produced during replication.

The alterations engineered into any of the viral enzymes include but arenot limited to insertions, deletions and substitutions in the amino acidsequence of the active site of the molecule. For example, the bindingsite of the enzyme could be altered so that its binding affinity forsubstrate is reduced, and as a result, the enzyme is less specificand/or efficient. For example, a target of choice is the viralpolymerase complex since temperature sensitive mutations exist in allpolymerase proteins. Thus, changes introduced into the amino acidpositions associated with such temperature sensitivity can be engineeredinto the viral polymerase gene so that an attenuated strain is produced.

5.4.1 The RSV L Gene as a Target for Attenuation

In accordance with the present invention, the RSV L gene is an importanttarget to generate recombinant RSV with an attenuated phenotype. The Lgene represents 48% of the entire RSV genome. The present inventionencompasses generating L gene mutants with defined mutations or randommutations in the RSV L gene. Any number of techniques known to thoseskilled in the art may be used to generate both defined or randommutations into the RSV L gene. Once the mutations have been introduced,the functionality of the L gene cDNA mutants are screened in vitro usinga minigenome replication system and the recovered L gene mutants arethen further analyzed in vitro and in vivo.

The following strategies are exemplary of the approaches which may beused to generate mutants with an attenuated phenotype. Further, thefollowing strategies as described below have been applied to the L geneonly by way of example and may also be applied to any of the other RSVgenes.

One approach to generate mutants with an attenuated phenotype utilizes ascanning mutagenesis approach to mutate clusters of charged amino acidsto alanines. This approach is particularly effective in targetingfunctional domains, since the clusters of charged amino acids generallyare not found buried within the protein structure. Replacing the chargedamino acids with conservative substitutions, such as neutral aminoacids, e.g., alanine, should not grossly alter the structure of theprotein but rather, should alter the activity of the functional domainof the protein. Thus, disruption of charged clusters should interferewith the ability of that protein to interact with other proteins, thusmaking the mutated protein's activity thermosensitive which can yieldtemperature sensitive mutants.

A cluster of charged amino acids may be arbitrarily defined as a stretchof five amino acids in which at least two or more residues are chargedresidues. In accordance with the scanning mutagenesis approach all ofthe charged residues in the cluster are mutated to alanines usingsite-directed mutagenesis. Due to the large site of the RSV L gene,there are many clustered charged residues. Within the L gene, there areat least two clusters of four contiguous charged residues and at leastseventeen clusters of three contiguous charged residues. At least two tofour of the charged residues in each cluster may be substituted with aneutral amino acid, e.g., alanine.

In yet another approach to generate mutants with an attenuated phenotypeutilizes a scanning mutagenesis approach to mutate cysteines to aminoacids, such as glycines or alanines. Such an approach takes advantage ofthe frequent role of cysteines in intramolecular and intermolecular bondformations, thus by mutating cysteines to another residue, such as aconservative substitution e.g., valine or alanine, or a drasticsubstitution e.g., aspartic acid, the stability and function of aprotein may be altered due to disruption of the protein's tertiarystructure. There are approximately thirty-nine cysteine residues presentin the RSV L gene.

In yet another approach random mutagenesis of the RSV L gene will coverresidues other than charged or cysteines. Since the RSV L gene is verylarge, such an approach may be accomplished by mutagenizing large cDNAfragments of the L gene by PCR mutagenesis. The functionality of suchmutants may be screened by a minigenome replication system and therecovered mutants are then further analyzed in vitro and in vivo.

5.5 Vaccine Formulations Using the Chimeric Viruses

Virtually any heterologous gene sequence may be constructed into thechimeric viruses of the invention for use in vaccines. In a preferredembodiment, the present invention relates to bivalent RSV vaccines whichconfers protection against RSV-A and RSV-B. To formulate such a vaccine,a chimeric RS virus is used which expresses the antigenic polypeptidesof both RSV-A and RSV-B subtypes. In yet another preferred embodiment,the present invention relates to a bivalent vaccine which confersprotection against both RSV and influenza. To formulate such a vaccine,a chimeric RS virus is used which expresses the antigenic polypeptidesof both RSV and influenza.

Preferably, epitopes that induce a protective immune response to any ofa variety of pathogens, or antigens that bind neutralizing antibodiesmay be expressed by or as part of the chimeric viruses. For example,heterologous gene sequences that can be constructed into the chimericviruses of the invention for use in vaccines include but are not limitedto sequences derived from a human immunodeficiency virus (HIV),preferably type 1 or type 2. In a preferred embodiment, an immunogenicHIV-derived peptide which may be the source of an antigen may beconstructed into a chimeric influenza virus that may then be used toelicit a vertebrate immune response.

Such HIV-derived peptides may include, but are not limited to sequencesderived from the env gene (i.e., sequences encoding all or part ofgp160, gp120, and/or gp41), the pol gene (i.e., sequences encoding allor part of reverse transcriptase, endonuclease, protease, and/orintegrase), the gag gene (i.e., sequences encoding all or part of p7,p6, p55, p17/18, p24/25), tat, rev, nef, vif, vpu, vpr, and/or vpx.

Other heterologous sequences may be derived from hepatitis B virussurface antigen (HBsAg); the glycoproteins of herpes virus (e.g. gD,gE); VP1 of poliovirus; antigenic determinants of non-viral pathogenssuch as bacteria and parasites, to name but a few. In anotherembodiment, all or portions of immunoglobulin genes may be expressed.For example, variable regions of anti-idiotypic immunoglobulins thatmimic such epitopes may be constructed into the chimeric viruses of theinvention.

Either a live recombinant viral vaccine or an inactivated recombinantviral vaccine can be formulated. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

In this regard, the use of genetically engineered RSV (vectors) forvaccine purposes may require the presence of attenuation characteristicsin these strains. Current live virus vaccine candidates for use inhumans are either cold adapted, temperature sensitive, or passaged sothat they derive several (six) genes from avian viruses, which resultsin attenuation. The introduction of appropriate mutations (e.g.,deletions) into the templates used for transfection may provide thenovel viruses with attenuation characteristics. For example, specificmissense mutations which are associated with temperature sensitivity orcold adaption can be made into deletion mutations. These mutationsshould be more stable than the point mutations associated with cold ortemperature-sensitive mutants and reversion frequencies should beextremely low.

Alternatively, chimeric viruses with “suicide” characteristics may beconstructed. Such viruses would go through only one or a few rounds ofreplication in the host. When used as a vaccine, the recombinant viruswould go through a single replication cycle and induce a sufficientlevel of immune response but it would not go further in the human hostand cause disease. Recombinant viruses lacking one or more of theessential RS virus genes would not be able to undergo successive roundsof replication. Such defective viruses can be produced byco-transfecting reconstituted RNPs lacking a specific gene(s) into celllines which permanently express this gene(s). Viruses lacking anessential gene(s) will be replicated in these cell lines but whenadministered to the human host will not be able to complete a round ofreplication. Such preparations may transcribe and translate—in thisabortive cycle—a sufficient number of genes to induce an immuneresponse. Alternatively, larger quantities of the strains could beadministered, so that these preparations serve as inactivated (killed)virus vaccines. For inactivated vaccines, it is preferred that theheterologous gene product be expressed as a viral component, so that thegene product is associated with the virion. The advantage of suchpreparations is that they contain native proteins and do not undergoinactivation by treatment with formalin or other agents used in themanufacturing of killed virus vaccines.

In another embodiment of this aspect of the invention, inactivatedvaccine formulations may be prepared using conventional techniques to“kill” the chimeric viruses. Inactivated vaccines are “dead” in thesense that their infectivity has been destroyed. Ideally, theinfectivity of the virus is destroyed without affecting itsimmunogenicity. In order to prepare inactivated vaccines, the chimericvirus may be grown in cell culture or in the allantois of the chickembryo, purified by zonal ultracentrifugation, inactivated byformaldehyde or β-propiolactone, and pooled. The resulting vaccine isusually inoculated intramuscularly.

Inactivated viruses may be formulated with a suitable adjuvant in orderto enhance the immunological response. Such adjuvants may include butare not limited to mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions;peptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum.

Many methods may be used to introduce the vaccine formulations describedabove, these include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. It may be preferable to introduce the chimeric virusvaccine formulation via the natural route of infection of the pathogenfor which the vaccine is designed. Where a live chimeric virus vaccinepreparation is used, it may be preferable to introduce the formulationvia the natural route of infection for influenza virus. The ability ofRSV and influenza virus to induce a vigorous secretory and cellularimmune response can be used advantageously. For example, infection ofthe respiratory tract by chimeric RSV or influenza viruses may induce astrong secretory immune response, for example in the urogenital system,with concomitant protection against a particular disease causing agent.

The following sections describe by way of example, and not bylimitation, the manipulation of the negative strand RNA viral genomesusing RSV as an example to demonstrate the applicability of the methodsof the present invention to generate chimeric viruses for the purposesof heterologous gene expression, generating infectious viral particlesand attenuated viral particles for the purposes of vaccination.

6. RESCUE OF INFECTIOUS RESPIRATORY SYNCYTIAL VIRUSES (RSV) USING RNADERIVED FROM SPECIFIC RECOMBINANT DNAS

This example describes a process for the rescue of infectiousrespiratory syncytial virus (RSV), derived from recombinant cDNAsencoding the entire RSV RNA genome into stable and infectious RSVs, asnoted in Section 5 above. The method described may be applied to bothsegmented and non-segmented RNA viruses, including orthomyxovirus,paramyxovirus, e.g., Sendai virus, parainfluenza virus types 1-4, mumps,newcastle disease virus; morbillivirus, e.g., measles, canine distempervirus, rinderpest virus; pneumovirus, e.g., respiratory syncytial virus;rhabdovirus, e.g., rabies, vesiculovirus, vesicular stomatitis virus;but is described by way of example in terms of RSV. This process can beused in the production of chimeric RSV viruses which can express foreigngenes, i.e., genes non-native to RSV, including other viral proteinssuch as the HIV env protein. Another exemplary way to achieve theproduction of chimeric RSV involves modifying existing, native RSVgenes, as is further described. Accordingly, this example also describesthe utility of this process in the directed attenuation of RSVpathogenicity, resulting in production of a vaccine with defined,engineered biological properties for use in humans.

The first step of the rescue process involving the entire RSV RNA genomerequires synthesis of a full length copy of the 15 kilobase (Kb) genomeof RSV strain A2. This is accomplished by splicing together subgenomicdouble strand cDNAs (using standard procedures for genetic manipulation)ranging in size from 1 kb-3.5 kb, to form the complete genomic cDNA.Determination of the nucleotide sequence of the genomic cDNA allowsidentification of errors introduced during the assembly process; errorscan be corrected by site directed mutagenesis, or by substitution of theerror region with a piece of chemically synthesized double strand DNA.Following assembly, the genomic cDNA is positioned adjacent to atranscriptional promoter (e.g., the T7 promoter) at one end and DNAsequence which allows transcriptional termination at the other end,e.g., a specific endonuclease or a ribozyme, to allow synthesis of aplus or minus sense RNA copy of the complete virus genome in vitro or incultured cells. The leader or trailer sequences may contain additionalsequences as desired, such as flanking ribozyme and tandem T7transcriptional terminators. The ribozyme can be a hepatitis delta virusribozyme or a hammerhead ribozyme and functions to yield an exact 3′ endfree of non-viral nucleotides.

In accordance with this aspect of the invention, mutations,substitutions or deletions can be made to the native RSV genomicsequence which results in an increase in RSV promoter activity.Applicants have demonstrated that even an increase in RSV promoteractivity greatly enhances the efficiency of rescue of RSV, allowing forthe rescue of infectious RSV particles from a full-length RSV cDNAcarrying the mutation. In particular, a point mutation at position 4 ofthe genome (C to G) results in a several fold increase in promoteractivity and the rescue of infectious viral particles from a full-lengthRSV cDNA clone carrying the mutation.

The rescue process utilizes the interaction of full-length RSV strain A2genome RNA, which is transcribed from the constructed cDNA, with helperRSV subgroup B virus proteins inside cultured cells. This can beaccomplished in a number of ways. For example, full-length virus genomicRNA from RSV strain A2 can be transcribed in vitro and transfected intoRSV strain B9320 infected cells, such as 293 cells using standardtransfection protocols. In addition, in vitro transcribed genomic RNAfrom RSV strain A2 can be transfected into a cell line expressing theessential RSV strain A2 proteins (in the absence of helper virus) fromstably integrated virus genes.

Alternatively, in vitro transcribed virus genome RNA (RSV strain A2) canalso be transfected into cells infected with a heterologous virus (e.g.,in particular vaccinia virus) expressing the essential helper RSV strainA2 proteins, specifically the N, P, L and/or M2-ORF1 proteins. Inaddition the in vitro transcribed genomic RNA may be transfected intocells infected with a heterologous virus, for example vaccinia virus,expressing T7 polymerase, which enables expression of helper proteinsfrom transfected plasmid DNAs containing the helper N, P, L and M2/ORF1genes.

As an alternative to transfection of in vitro transcribed genomic RNA,plasmid DNA containing the entire RSV cDNA construct may be transfectedinto cells infected with a heterologous virus, for example vacciniavirus, expressing the essential helper RSV strain A2 proteins and T7polymerase, thereby enabling transcription of the entire RSV genomic RNAfrom the plasmid DNA containing the RSV cDNA construct. The vacciniavirus need not however, supply the helper proteins themselves but onlythe T7 polymerase; then helper proteins may be expressed fromtransfected plasmids containing the RSV N, P, L and M2/ORF1 genes,appropriately positioned adjacent to their own T7 promoters.

When replicating virus is providing the helper function during rescueexperiments, the B9320 strain of RSV is used, allowing differentiationof progeny rescue directed against RSV B9320. Rescued RSV strain A2 ispositively identified by the presence of specific nucleotide ‘marker’sequences inserted in the cDNA copy of the RSV genome prior to rescue.

The establishment of a rescue system for native, i.e., ‘wild-type’ RSVstrain A2 allows modifications to be introduced into the cDNA copy ofthe RSV genome to construct chimeric RSV containing sequencesheterologous in some manner to that of native RSV, such that theresulting rescued virus may be attenuated in pathogenicity to provide asafe and efficacious human vaccine as discussed in Section 5.4 above.The genetic alterations required to cause virus attenuation may be gross(e.g., translocation of whole genes and/or regulatory sequences withinthe virus genome), or minor (e.g., single or multiple nucleotidesubstitution(s), addition(s) and/or deletion(s) in key regulatory orfunctional domains within the virus genome), as further described indetail.

In addition to alteration(s) (including alteration resulting fromtranslocation) of the RSV genetic material to provide heterologoussequence, this process permits the insertion of ‘foreign’ genes (i.e.,genes non-native to RSV) or genetic components thereof exhibitingbiological function or antigenicity in such a way as to give expressionof these genetic elements; in this way the modified, chimeric RSV canact as an expression system for other heterologous proteins or geneticelements, such as ribozymes, anti-sense RNA, specificoligoribonucleotides, with prophylactic or therapeutic potential, orother viral proteins for vaccine purposes.

6.1 Rescue of the Leader and Trailer Sequences of RSV Strain A2 UsingRSV Strain B9320 as Helper Virus

6.1.1 Viruses and Cells

Although RSV strain A2 and RSV strain B9320 were used in this Example,they are exemplary. It is within the skill in the art to use otherstrains of RSV subgroup A and RSV subgroup B viruses in accordance withthe teachings of this Example. Methods which employ such other strainsare encompassed by the invention.

RSV strain A2 and RSV strain B9320 were grown in Hep-2 cells and Verocells respectively, and 293 cells were used as host duringtransfection/rescue experiments. All three cell lines were obtained fromthe ATCC (Rockville, Md.).

6.1.2 Construction & Functional Analysis of Reporter Plasmids

Plasmid pRSVA2CAT (FIG. 1) was constructed as described below.

The cDNAs of the 44 nucleotide leader and 155 nucleotide trailercomponents of RSV strain A2 (see Mink et al., Virology 185:615-624(1991); Collins et al., Proc. Natl. Acad. Sci. 88:9663-9667 (1991)), thetrailer component also including the promoter consensus sequence ofbacteriophage T7 polymerase, were separately assembled by controlledannealing of oligonucleotides with partial overlapping complementarity(see FIG. 1). The oligonucleotides used in the annealing weresynthesized on an Applied Biosystems DNA synthesizer (Foster City,Calif.). The separate oligonucleotides and their relative positions inthe leader and trailer sequences are indicated in FIG. 1. Theoligonucleotides used to construct the leader were: 1. 5′CGA CGC ATA TTACGC GAA AAA ATG (SEQ ID NO:1) CGT ACA ACA AAC TTG CAT AAA C 2. 5′CAA AAAAAT GGG GCA AAT AAG AAT (SEQ ID NO:2) TTG ATA AGT ACC ACT TAA ATT TAA CT3. 5′CTA GAG TTA AAT TTA AGT GGT ACT (SEQ ID NO:3) 4. 5′TAT CAA ATT CTTATT TGC CCC ATT (SEQ ID NO:4) TTT TTG GTT TAT GCA AGT TTG TTG TA 5.5′CGC ATT TTT TCG CGT AAT ATG CGT (SEQ ID NO:5) CGG TAC

The oligonucleotides used to construct the trailer were: 1. 5′GTA TTCAAT TAT AGT TAT TAA (SEQ ID NO:6) AAA TTA AAA ATC ATA TAA TTT TTT AAA TA2. 5′ACT TTT AGT GAA CTA ATC CTA (SEQ ID NO:7) AAG TTA TCA TTT TAA TCTTGG AGG AAT AA 3. 5′ATT TAA ACC CTA ATC TAA TTG (SEQ ID NO:8) GTT TATATG TGT ATT AAC TAA ATT ACG AG 4. 5′ATA TTA GTT TTT GAC ACT TTT (SEQ IDNO:9) TTT CTC GTT ATA GTG AGT CGT ATT A 5. 5′AGC TTA ATA CGA CTC ACT ATA(SEQ ID NO:10) ACG A 6. 5′GAA AAA AAG TGT CAA AAA CTA (SEQ ID NO:11) ATATCT CGT AAT TTA GTT AAT ACA CAT AT 7. 5′AAA CCA ATT AGA TTA GGG TTT (SEQID NO:12) AAA TTT ATT CCT CCA AGA TTA AAA TGA TA 8. 5′ACT TTA GGA TTAGTT CAC TAA (SEQ ID NO:13) AAG TTA TTT AAA AAA TTA TAT GAT TTT TA 9.5′ATT TTT AAT AAC TAT AAT TGA (SEQ ID NO:14) ATA CTG CA

The complete leader and trailer cDNAs were then ligated to thechloramphenicol-acetyl-transferase (CAT) reporter gene XbaI and PstIsites respectively to form a linear −1 kb RSV/CAT cDNA construct. ThiscDNA construct was then ligated into the Kpn I and Hind II sites ofpUC19. The integrity of the final pRSVA2CAT construct was checked by gelanalysis for the size of the Xba I/Pst I and Kpn I/Hind II digestionproducts. The complete leader and trailer cDNAs were also ligated to thegreen fluorescent protein (GFP) gene using appropriate restrictionenzyme sites to form a linear cDNA construct. The resulting RSV-GFP-CATis a bicistronic reporter construct which expresses both CAT and GFP.

In vitro transcription of Hga I linearized pRSVA2CAT with bacteriophageT7 polymerase was performed according to the T7 supplier protocol(Promega Corporation, Madison, Wis.). Confluent 293 cells in six-welldishes (−1×10⁶ cells per well) were infected with RSV strain B9320 at 1plaque forming units (p.f.u.) per cell and 1 hour later were transfectedwith 5-10 μg of the in vitro transcribed RNA from the pRSVA2CATconstruct. The transfection procedure followed the transfectionprocedure of Collins et al., Virology 195:252-256 (1993) and employedTransect/ACT™ and Opti-MEM reagents according to the manufacturersspecifications (Gibco-BRL, Bethesda, Md.). At 24 hours post-infectionthe 293 cells were assayed for CAT activity using a standard protocol(Current Protocols in Molecular Biology, Vol. 1, Chapter 9.6.2; Gorman,et al., 1982) Mol. Cell Biol. 2: 1044-1051). The detection of highlevels of CAT activity indicated that in vitro transcribed negativesense RNA containing the ‘leader’ and ‘trailer’ regions of the RSV A2strain genome and the CAT gene can be encapsidated, replicated andexpressed using proteins supplied by RSV strain B9320 (See FIG. 2). Thelevel of CAT activity observed in these experiments was at least as highas that observed in similar rescue experiments where homologous RSVstrain A2 was used as helper virus. The ability of an antigenicallydistinct subgroup B RSV strain B9320 to support the encapsidation,replication and transcription of a subgroup A RSV strain A2 RNA has toour knowledge hitherto not been formally reported.

6.2 Construction of a cDNA Representing the Complete Genome of RSV

To obtain a template for cDNA synthesis, RSV genomic RNA, comprising15,222 nucleotides, was purified from infected Hep-2 cells according tothe method described by Ward et al., J. Gen. Virol. 64:167-1876 (1983).Based on the published nucleotide sequence of RSV, oligonucleotides weresynthesized using an Applied Biosystems DNA synthesizer (AppliedBiosystems, Foster City, Calif.) to act as primers for first and secondstrand cDNA synthesis from the genomic RNA template. The nucleotidesequences and the relative positions of the cDNA primers and keyendonuclease sites within the RSV genome are indicated in FIG. 3. Theproduction of cDNAs from virus genomic RNA was carried out according tothe reverse transcription/polymerase chain reaction (RT/PCR) protocol ofPerkin Elmer Corporation, Norwalk, Conn. (see also Wang et al., (1989)Proc. Natl. Acad. Sci. 86:9717-9721); the amplified cDNAs were purifiedby electroelution of the appropriate DNA band from agarose gels.Purified DNA was ligated directly into the pCRII plasmid vector(Invitrogen Corp. San Diego), and transformed into either ‘One Shot E.coli cells (Invitrogen) or ‘SURE’ E. coli cells (Stratagene, San Diego).The resulting, cloned, virus specific, cDNAs were assembled by standardcloning techniques (Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor laboratory Press (Cold Spring Harbor, N.Y.,1989) to produce a cDNA spanning the complete RSV genome. The entirecDNA genome was sequenced, and incorrect sequences were replaced byeither site-directed mutagenesis or chemically synthesized DNA.Nucleotide substitutions were introduced at bases 7291 and 7294 (withbase number 1 being at the start of the genomic RNA 3′ end) in the ‘F’gene, to produce a novel Stu I endonuclease site, and at positions 7423,7424, and 7425 (also in the F gene) to produce a novel Pme I site. Thesechanges were designed to act as definitive markers for rescue events.The bacteriophage T7 polymerase and the Hga I endonuclease site wereplaced at opposite ends of the virus genome cDNA such that eithernegative or positive sense virus genome RNA can be synthesized in vitro.The cDNAs representing the T7 polymerase promoter sequence and therecognition sequence for Hga I were synthesized on an Applied BiosystemsDNA synthesizer and were separately ligated to the ends of the virusgenome cDNA, or were added as an integral part of PCR primers duringamplification of the terminal portion of the genome cDNA, whereappropriate; the latter procedure was used when suitable endonucleasesites near the genome cDNA termini were absent, preventing directligation of chemically synthesized T7 promoter/Hga I site cDNA to thegenome cDNA. This complete construct (genome cDNA and flanking T7promoter/Hga I recognition sequence) was then cloned into the Kpn I/NotI sites of the Bluescript II SK phagemid (Stratagene, San Diego) fromwhich the endogenous T7 promoter has been removed by site-directedmutagenesis. RNA transcribed from this complete genome construct may berescued using RSV subgroup B helper virus to give infectious RSV inaccordance with Example 6.1. This basic rescue system for the completenative, i.e., ‘wild-type’ RSV A2 strain genomic RNA can be employed tointroduce a variety of modifications into the cDNA copy of the genomeresulting in the introduction of heterologous sequences into the genome.Such changes can be designed to reduce viral pathogenicity withoutrestricting virus replication to a point where rescue becomes impossibleor where virus gene expression is insufficient to stimulate adequateimmunity.

The following oligonucleotides were used to construct the ribozyme/T7terminator sequence: 5′GGT*GGCCGGCATGGTCCCAGC (SEQ ID NO:31)3′CCA CCGGCCGTACCAGGGTCG CTCGCTGGCGCCGGCTGGGCAACA (SEQ ID NO:32)GAGCGACCGCGGCCGACCCGTGTG TTCCGAGGGGACCGTCCCCTCGGT (SEQ ID NO:33)AAGGCTCCCCTGGCAGGGGAGCCA AATGGCGAATGGGACGTCGACAGC (SEQ ID NO:34)TTACCGCTTACCCTGCAGCTGTCG TAACAAAGCCCGAAGGAAGCT (SEQ ID NO:35)ATTGTTTCGGGCTTCCTTCGA GAGTTGCTGCTGCCACCGTTG (SEQ ID NO:36)CTCAACGACGACGGAGGCAAC AGCAATAACTAGATAACCTTGGG (SEQ ID NO:37)TCGTTATTGATCTATTGGAACCC CCTCTAAACGGGTCTTGAGGGTCT (SEQ ID NO:38)GGAGATTTGCCCAGAACTCCCAGA TTTTGCTGAAAGGAGGAACTA (SEQ ID NO:39)AAAACGACTTTCCTCCTTGAT TATGCGGCCGCGTCGACGGTA (SEQ ID NO:40)ATACGCCGGCGEAGCTGCCAT CCGGGCCCGCCTTCGAAG3′ (SEQ ID NO:41)GGCCCGGGCGGAAGCTTC5′

A cDNA clone containing the complete genome of RSV a T7 promoter, ahepatitis delta virus ribozyme and a T7 terminator was generated. Thisconstruct can be used to generate antigenomic RNA or RSV in vivo in thepresence of T7 polymerase. Sequence analysis indicated that the plasmidcontained few mutations in RSV genome.

6.2.1 Modifications of the RSV Genome

Modifications of the RSV RNA genome can comprise gross alterations ofthe genetic structure of RSV, such as gene shuffling. For example, theRSV M1 gene can be translocated to a position closer to the 5′ end ofthe genome, in order to take advantage of the known 3′ to 5′ gradient invirus gene expression, resulting in reduced levels of M1 proteinexpression in infected cells and thereby reducing the rate of virusassembly and maturation. Other genes and/or regulatory regions may alsobe translocated appropriately, in some cases from other strains of RSVof human or animal origin. For example, the F gene (and possibly the ‘G’gene) of the human subgroup B RSV could be inserted into an otherwiseRSV strain A genome (in place or, or in addition to the RSV strain A Fand G genes).

In another approach, the RNA sequence of the RSV viruses N protein canbe translocated from its 3′ proximal site to a position closer to the 5′end of the genome, again taking advantage of the 3′ to 5′ gradient ingene transcription to reduce the level of N protein produced. Byreducing the level of N protein produced, there would result aconcomitant increase in the relative rates of transcription of genesinvolved in stimulating host immunity to RSV and a concomitant reductionin the relative rate of genome replication. Thus, by translocating theRSV RNA sequence coding for RSV N protein, a chimeric RS virus havingattenuated pathogenicity relative to native RSV will be produced.

Another exemplary translocation modification resulting in the productionof attenuated chimeric RSV comprises the translocation of the RSV RNAsequence coding for the L protein of RSV. This sequence of the RS virusis believed responsible for viral polymerase protein production. Bytranslocating the RSV sequence coding for L protein from its native 5′terminal location in the native RSV genome to a location at or near the3′ terminus of the genome, a chimeric RSV virus exhibiting attenuatedpathogenicity will be produced. Yet another exemplary translocationcomprises the switching the locations of the RSV RNA sequences codingfor the RSV G and F proteins (i.e., relative to each other in thegenome) to achieve a chimeric RSV having attenuated pathogenicityresulting from the slight modification in the amount of the G and Fproteins produced. Such gene shuffling modifications as are exemplifiedand discussed above are believed to result in a chimeric, modified RSVhaving attenuated pathogenicity in comparison to the native RSV startingmaterial. The nucleotide sequences for the foregoing encoded proteinsare known, as is the nucleotide sequence for the entire RSV genome. SeeMcIntosh, Respiratory Syncytial Virus in Virology, 2d Ed. edited by B.N. Fields, D. M. Knipe et al., Raven Press, Ltd. New York, 1990 Chapter38, pp 1045-1073, and references cited therein.

These modifications can additionally or alternatively compriselocalized, site specific, single or multiple, nucleotide substitutions,deletions or additions within genes and/or regulatory domains of the RSVgenome. Such site specific, single or multiple, substitutions, deletionsor additions can reduce virus pathogenicity without overly attenuatingit, for example, by reducing the number of lysine or arginine residuesat the cleavage site in the F protein to reduce efficiency of itscleavage by host cell protease (which cleavage is believed to be anessential step in functional activation of the F protein), and therebypossibly reduce virulence. Site specific modifications in the 3′ or 5′regulatory regions of the RSV genome may also be used to increasetranscription at the expense of genome replication. In addition,localized manipulation of domains within the N protein, which isbelieved to control the switch between transcription and replication canbe made to reduce genome replication but still allow high levels oftranscription. Further, the cytoplasmic domain(s) of the G and Fglycoproteins can be altered in order to reduce their rate of migrationthrough the endoplasmic reticulum and golgi of infected cells, therebyslowing virus maturation. In such cases, it may be sufficient to modifythe migration of G protein only, which would then allow additionalup-regulation of ‘F’ production, the main antigen involved instimulating neutralizing antibody production during RSV infections. Suchlocalized substitutions, deletions or additions within genes and/orregulatory domains of the RSV genome are believed to result in chimeric,modified RSV also having reduced pathogenicity relative to the nativeRSV genome.

6.3 Rescue of a cDNA Representing the Complete Genome of RSV

6.3.1 The Construction and Functional Analysis of Expression Plasmids

The RSV, N, P, and L genes encode the viral polymerase of RSV. Thefunction of the RSV M genes is unknown. The ability of RSV, N, P, M, andL expression plasmids to serve the function of helper RSV strain AZproteins was assessed as described below. The RSV, N, P, L, and M2-1genes were cloned into the modified PCITE 2a(+) vector (Novagen,Madison, Wis.) under the control of the T7 promoter and flanked by a T7terminator at it's 3′ end. PCITE-2a(+) was modified by insertion of a T7terminator sequence from PCITE-3a(+) into the Alwn I and Bgl II sites ofpCITE-2a(+). The functionality of the N, P, and L expression plasmidswas determined by their ability to replicate the transfected pRSVA2CAT.At approximately 80% confluency, Hep-2 cells in six-well plates wereinfected with MVA at a moi of 5. After 1 hour, the infected cells weretransfected with pRSVA2CAT (0.5 mg), and plasmids encoding the N (0.4mg), P (0.4 mg), and L (0.2 mg) genes using lipofecTACE (LifeTechnologies, Gaithersburg, Md.). The transfection proceeded for 5 hoursor overnight and then the transfection medium was replaced with freshMEM containing 2% (fetal bovine serum) FBS. Two days post-infection, thecells were lysed and the lysates were analyzed for CAT activity usingBoehringer Mannheim's CAT ELISA kit. CAT activity was detected in cellsthat had been transfected with N, P, and L plasmids together withPRSVAZCAT. However, no CAT activity was detected when any one of theexpression plasmids was omitted. Furthermore, co-transfection ofRSV-GFP-CAT with the N, P, and L expression plasmids resulted inexpression of both GFP and CAT proteins. The ratios of differentexpression plasmids and moi of the recombinant vaccinia virus wereoptimized in the reporter gene expression system.

6.3.2 Recovery of Infectious RSV from the Complete RSV cDNA

Hep-2 cells were infected with MVA (recombinant vaccinia virusexpressing T7 polymerase) at an moi of one. Fifty minutes later,transfection mixture was added onto the cells. The transfection mixtureconsisted of 2 μg of N expression vector, 2 μg of P expression vector, 1μg of L expression vector, 1.25 μg of M2/ORF1 expression vector, 2 μg ofRSV genome clone with enhanced promoter, 50 μl of LipofecTACE (LifeTechnologies, Gaithersburg, Md.) and 1 ml OPTI-MEM. One day later, thetransfection mixture was replaced by MEM containing 2% FCS. The cellswere incubated at 37° C. for 2 days. The transfection supernatant washarvested and used to infect fresh Hep-2 cells in the presence of 40μg/ml arac (drug against vaccinia virus). The infected Hep2 cells wereincubated for 7 days. After harvesting the P1 supernatant, cells wereused for immunostaining using antibodies directed against F protein ofRSV A2 strain. Six positively stained loci with visible cell-cell-fusion(typical for RSV infection) were identified. The RNA was extracted fromPI supernatant, and used as template for RT-PCR analysis. PCR productscorresponding to F and M2 regions were generated. both productscontained the introduced markers. In control, PCR products derived fromnatural RSV virus lacked the markers.

A point mutation was created at position 4 of the leader sequence of theRSV genome clone (C residue to G) and this genome clone was designatedpRSVC4GLwt. This clone has been shown in a reporter gene context toincrease the promoter activity by several fold compared to wild-type.After introduction of this mutation into the full-length genome,infectious virus was rescued from the cDNA clone. The rescuedrecombinant RSV virus formed smaller plaques than the wild-type RSVvirus (FIG. 8).

This system allows the rescue mutated RSV. Therefore, it may be anexcellent tool to engineer live-attenuated vaccines against RSV and touse RSV vector and viruses to achieve heterologous gene expression. Itmay be possible to express G protein of type B RSV into the type Abackground, so the vaccine is capable of protect both type A and type BRSV infection. It may also be possible to achieve attenuation andtemperature sensitive mutations into the RSV genome, by changing thegene order or by site-directed mutagenesis of the L protein.

6.4 Use of Monoclonal Antibodies to Differentiate Rescued Virus FromHelper Virus

In order to neutralize the RSV strain B9320 helper virus and facilitateidentification of rescued A 2 strain RSV, monoclonal antibodies againstRSV strain B9320 were made as follows.

Six BALB/c female mice were infected intranasally (i.n.) with 10⁵ plaqueforming units (p.f.u.) of RSV B9320, followed 5 weeks later byintraperitoneal (i.p.) inoculation with 10⁶-10⁷ pfu of RSV B9320 in amixture containing 50% complete Freund's adjuvant. Two weeks after i.p.inoculation, a blood sample from each mouse was tested for the presenceof RSV specific antibody using a standard neutralization assay (Beelerand Coelingh, J. Virol. 63:2941-2950 (1988)). Mice producing the highestlevel of neutralizing antibody were then further boosted with 10⁶ p.f.u.of RSV strain B9320 in phosphate buffered saline (PBS), injectedintravenously at the base of the tail. Three days later, the mice weresacrificed and their spleens collected as a source of monoclonalantibody producing B-cells. Splenocytes (including B-cells) were teasedfrom the mouse spleen through incisions made in the spleen capsule into5 ml of Dulbecco's Modified Eagle's Medium (DME). Clumps of cells wereallowed to settle out, and the remaining suspended cells were separatelycollected by centrifugation at 2000×g for 5 minutes at room temperature.These cell pellets were resuspended in 15 ml 0.83 (W/V) NH₄Cl, andallowed to stand for 5 minutes to lyse red blood cells. Splenocytes werethen collected by centrifugation as before through a 10 ml; cushion offetal calf serum. The splenocytes were then rinsed in DME, repelletedand finally resuspended in 20 ml of fresh DME. These splenocytes werethen mixed with Sp2/0 cells (a mouse myeloma cell line used as fusionpartners for the immortalization of splenocytes) in a ratio of 10:1,spleen cells: Sp2/0 cells. Sp2/0 cells were obtained from the ATCC andmaintained in DME supplemented with 10% fetal bovine serum. The cellmixture was then centrifuged for 8 minutes at 2000×g at roomtemperature. The cell pellet was resuspended in 1 ml of 50% polyethyleneglycol 1000 mol. wt. (PEG 1000), followed by addition of equal volumesof DME at 1 minute intervals until a final volume of 25 ml was attained.The fused cells were then pelleted as before and resuspended at 3.5×10⁶spleen cells ml in growth medium (50% conditioned medium from SP2/0cells, 50% HA medium containing 100 ml RPMI 25 ml F.C.S., 100 μgmlgentamicin, 4 ml 50× Hypoxanthine, Thymidine, Aminopterin (HAT) mediumsupplied as a prepared mixture of Sigma Chem. Co., St. Louis, Mo.). Thecell suspension was distributed over well plates (200 μl well⁻¹) andincubated at 37° C., 95 humidity and 5% CO₂. Colonies of hybridoma cells(fused splenocytes and Sp2/0 cells) were then subcultured into 24 wellplates and grown until nearly confluent; the supernatant growth mediumwas then sampled for the presence of RSV strain B9320 neutralizingmonoclonal antibody, using a standard neutralization assay (Beeler andCoelingh, J. Virol. 63:2941-50 (1988)). Hybridoma cells from wells withneutralizing activity were resuspended in growth medium and diluted togive a cell density of 0.5 cells per 100 μl and plated out in 96 wellplates, 200 μl per well. This procedure ensured the production ofmonoclones (i.e. hybridoma cell lines derived from a single cell) whichwere then reassayed for the production of neutralizing monoclonalantibody. Those hybridoma cell lines which produced monoclonal antibodycapable of neutralizing RSV strain B9320 but not RSV strain A2 weresubsequently infected into mice, i.p. (10⁶ cells per mouse). Two weeksafter the i.p. injection mouse ascites fluid containing neutralizingmonoclonal antibody for RSV strain B9320 was tapped with a 19 gaugeneedle, and stored at −20° C.

This monoclonal antibody was used to neutralize the RSV strain B9320helper virus following rescue of RSV strain A2 as described in Section9.1. This was carried out by diluting neutralizing monoclonal antibody 1in 50 with molten 0.4% (w/v) agar in Eagle's Minimal Essential Medium(EMEM) containing 1% F.C.S. This mixture was then added to Hep-2 cellmonolayers, which had been infected with the progeny of rescueexperiments at an m.o.i. of 0.1-0.01 p.f.u. per cell. The monoclonalantibody in the agar overlay inhibited the growth of RSV strain B9320,but allowed the growth of RSV strain A2, resulting in plaque formationby the A2 strain. These plaques were picked using a pasteur pipette toremove a plug a agar above the plaque and the infected cells within theplaque; the cells and agar plug were resuspended in 2 ml of EMEM, 1%FCS, and released virus was plaqued again in the presence of monoclonalantibody on a fresh Hep-2 cell monolayer to further purify from helpervirus. The twice plaqued virus was then used to infect Hep-2 cells in 24well plates, and the progeny from that were used to infect six-wellplates at an m.o.i. of 0.1 p.f.u. per cell. Finally, total infected cellRNA from one well of a six-well plates was used in a RT/PCR reactionusing first and second strand primers on either side of the ‘markersequences’ (introduced into the RSV strain A2 genome to act as a meansof recognizing rescue events) as described in Section 6.2 above. The DNAproduced from the RT/PCR reaction was subsequently digested with Stu Iand Pme I to positively identify the ‘marker sequences’ introduced intoRSV strain A2 cDNA, and hence to establish the validity of the rescueprocess.

7. RESCUE OF INFECTIOUS RSV PARTICLES IN THE ABSENCE OF M2 EXPRESSION

The following experiments were conducted to compare the efficiencies ofrescue of RS virions in the presence and absence of the M2/ORF1 gene. Ifthe M2/ORF1 gene function is not required to achieve rescue of RSVinfectious particles, it should be possible to rescue RS virions in theabsence of the expression of the M2/ORF1 gene function. In the presentanalysis, Hep-2 cells which are susceptible to RSV replication, wereco-transfected with plasmids encoding the ‘N’, ‘P’ and ‘L’ genes of theviral polymerase of RSV and the cDNA corresponding to the full-lengthantigenome of RSV, in the presence or absence of plasmid DNA encodingthe M2/ORF1 gene, and the number of RSV infectious units were measuredin order to determine whether or not the M2/ORF1 gene product wasrequired to rescue infectious RSV particles.

The following plasmids were used in the experiments described below: acDNA clone encoding the full-length antigenome of RSV strain A2,designated pRSVC4GLwt; and plasmids encoding the N, P, and L polymeraseproteins, and plasmid encoding the M2/ORF1 elongation factor, eachdownstream of a T7 RNA promoter, designated by the name of the viralprotein encoded.

pRSVC4GLwt was transfected, together with plasmids encoding proteins N,P and L, into Hep-2 cells which had been pre-infected with a recombinantvaccinia virus expressing the T7 RNA polymerase (designated MVA). Inanother set of Hep-2 cells, pRSVC4GLwt was co-transfected with plasmidsencoding the N, P and L polymerase proteins, and in addition a plasmidencoding the M2 function. Transfection and recovery of recombinant RSVwere performed as follows: Hep-2 cells were split in six-well dishes (35mm per well) 5 hours or 24 hours prior to transfection. Each wellcontained approximately 1×10⁶ cells which were grown in MEM (minimumessential medium) containing 10% FBS (fetal bovine serum). Monolayers ofHep-2 cells at 70%-80% confluence were infected with MVA at amultiplicity of infection (moi) of 5 and incubated at 35° C. for 60minutes. The cells were then washed once with OPTI-MEM (LifeTechnologies) and the medium of each dish replaced with 1 ml of OPTI-MEMand 0.2 ml of the transfection mixture. The transfection mixture wasprepared by mixing the four plasmids, pRSVC4GLwt, N, P and L plasmids ina final volume of 0.1 ml OPTI-MEM at amounts of 0.5-0.6 μg ofpRSVC4GLwt, 0.4 μg of N plasmid, 0.4 μg of P plasmid, and 0.2 μg of Lplasmid. A second mixture was prepared which additionally included 0.4μg M2/ORFI plasmid. The plasmid mixtures of 0.1 ml were combined with0.1 ml of OPTI-MEM containing 10 μl of lipofecTACE (Life Technologies,Gaithersburg, Md.) to constitute the complete transfection mixture.After a 15 minute incubation at room temperature, the transfectionmixture was added to the cells, and one day later this was replaced byMEM containing 2% FBS. Cultures were incubated at 35° C. for 3 days atwhich time the supernatants were harvested. Cells were incubated at 35°C. since the MVA virus is slightly temperature sensitive and is muchmore efficient at 35° C.

Three days post-transfection, the transfected cell supernatants wereassayed for the presence of RSV infectious units by an immunoassay whichwould indicate the presence of RSV packaged particles (see Table I). Inthis assay, 0.3-0.4 ml of the culture supernatants were passaged ontofresh (uninfected) Hep-2 cells and overlaid with 1% methylcellulose and1×L15 medium containing 2% FBS. After incubation for 6 days, thesupernatant was harvested and the cells were fixed and stained by anindirect horseradish peroxidase method, using a goat anti-RSV antibodywhich recognizes the RSV viral particle (Biogenesis, Sandown, N.H.)followed by a rabbit anti-goat antibody conjugated to horseradishperoxidase. The antibody complexes that bound to RSV-infected cells weredetected by the addition of a AEC-(3-amino-9-ethylcarbazole) chromogensubstrate (DAKO) according to the manufacturer's instructions. The RSVplaques were indicated by a black-brown coloration resulting from thereaction between the chromogen substrate and the RSV-antibody complexesbound to the plaques. The number of RSV plaques is expressed as thenumber of plaque forming units (p.f.u.) per 0.5 ml of transfectionsupernatant (see Table I).

Comparisons of the amount of RS virions recovered from the supernatantsof transfection dishes in the presence or absence of M2/ORF1 are shownin Table I. The results of four separate experiments demonstrated thatthe absence of M2/ORF1 from the transfection assay did not diminish thenumber of infectious units of RSV observed. Thus, the results of theseexperiments clearly indicate that RSV can be rescued in the absence ofthe M2/ORF1 from cells transfected only with plasmids encoding the threepolymerase proteins, N, P and L, and the cDNA encoding the full-lengthRSV antigenome. The rescue of true RS virions in the absence of M2/ORF1was further indicated by the ability to passage the rescued recombinantRSV for up to six passages. Therefore, the production of RSV virions isnot dependent on the expression of the M2/ORF1 gene, nor does theinclusion of the M2/ORF1 gene in the transfection assay increase theefficiency of true RSV rescue. TABLE I Production of infectious RSVthrough plasmid transfection is not dependent on expression of M20RF1Production of infectious RSV (pfu from 0.5 ml transfection supernatants)Expt. +M2 0RF1 −M2 ORF1 1. 6, 10(8) 16, 9(13) 2. 120, 46, 428(198) 100,122, 105(109) 3. 160, 180(170) 150, 133(142) 4. 588, 253, 725(522) 300,1000, 110(470)Each experiment was done singly, in duplicates or triplicates. Theaverage number of plaque forming units (pfu) from 0.5 ml transfectedcell supernatants is shown in the brackets.

cell supernatants is shown in the brackets.

8. EXAMPLE Expression of RSV Subgroup B-G and -F Proteins by RSV A2Strain

The following experiments were conducted to generate a chimeric RSVwhich expresses the antigenic polypeptides of more than one strain ofRSV. Two main antigenic subgroups (A and B) of respiratory syncytialvirus (RSV) cause human diseases. Glycoproteins F and G are the twomajor antigenic determinants of RSV. The F glycoproteins of subgroup Aand B viruses are estimated to be 50% related, while the relationship ofG glycoproteins is considerably less, about 1-5%. Infection of RSVsubgroup A induces either partial or no resistance to replication of asubgroup B strain and vice versa. Both subgroup A and subgroup B RSVvirus vaccines are needed to protect from RSV infection.

The first approach described herein is to make an infectious chimericRSV cDNA clone expressing subgroup B antigens by replacing the currentinfectious RSV A2 cDNA clone G and F region with subgroup B-G and -Fgenes. The chimeric RSV would be subgroup B antigenic specific. Thesecond approach described herein is to insert subgroup B-G gene in thecurrent A2 cDNA clone so that one virus would express both subgroup Aand B specific antigens.

8.1 Substitution of A2 G and F by B9320 G and F Genes

RSV subgroup B strain B9320 G and F genes were amplified from B9320 vRNAby RT/PCR and cloned into pCRII vector for sequence determination. BamHI site was created in the oligonucleotide primers used for RT/PCR inorder to clone the G and F genes from B9320 strain into A2 antigenomiccDNA (FIG. 4A). A cDNA fragment which contained G and F genes from 4326nt to 9387 nt of A2 strain was first subcloned into pUC19 (pUCR/H). BglII sites were created at positions of 4630 (SH/G intergenic junction)and 7554 (F/M2 intergenic junction), respectively by Quickchangesite-directed mutagenesis kit (Stratagene, Lo Jolla, Calif.). B9320 Gand F cDNA inserted in pCR.II vector was digested with BamH Irestriction enzyme and then subcloned into Bgl II digested pUCR/H whichhad the A2 G and F genes removed. The cDNA clone with A2 G and F genesreplaced by B9320 G and F was used to replace the Xho I to Msc I regionof the full-length A2 antigenomic cDNA. The resulting antigenomic cDNAclone was termed pRSVB-GF and was used to transfect Hep-2 cells togenerate infectious RSVB-GF virus.

Generation of chimeric RSVB-GF virus was as follows, pRSVB-GF wastransfected, together with plasmids encoding proteins N, P, L andM2/ORF1, into Hep-2 cells which had been infected with MVA, arecombinant vaccinia virus which expresses the T7 RNA polymerase. Hep-2cells were split a day before transfection in six-well dishes.Monolayers of Hep-2 cells at 60% -70% confluence were infected with MVAat moi of 5 and incubated at 35° C. for 60 min. The cells were thenwashed once with OPTI-MEM (Life Technologies, Gaithersburg, Md.). Eachdish was replaced with 1 ml of OPTI-MEM and added with 0.2 ml oftransfection medium. The transfection medium was prepared by mixing fiveplasmids in a final volume of 0.1 ml of OPTI-MEM medium, namely 0.6 μgof RSV antigenome pRSVB-GF, 0.4 μg of N plasmid, 0.4 μg of P plasmid,0.2 μg of L plasmid and 0.4 μg of M2/ORF1 plasmid. This was combinedwith 0.1 ml of OPTI-MEM containing 10 μl lipofecTACE (Life Technologies,Gaithersburg, Md. U.S.A.). After a 15 minute incubation at roomtemperature, the DNA/lipofecTACE was added to the cells and the mediumwas replaced one day later by MEM containing 2% FBS. Cultures werefurther incubated at 35° C. for 3 days and the supernatants harvested.Aliquots of culture supernatants (PO) were then used to infect freshHep-2 cells. After incubation for 6 days at 35° C., the supernatant washarvested and the cells were fixed and stained by an indirecthorseradish peroxidase method using goat anti-RSV antibody (Biogenesis,Sandown, N.H.) followed by a rabbit anti-goat antibody linked tohorseradish peroxidase. The virus infected cells were then detected byaddition of substrate chromogen (DAKO, Carpinteria, Calif., U.S.A.)according to the manufacturer's instructions. RSV-like plaques weredetected in the cells which were infected with the supernatants fromcells transfected with pRSVB-GF. The virus was further plaque purifiedtwice and amplified in Hep-2 cells.

Recombinant RSVB-GF virus was characterized by RT/PCR using RSV subgroupB specific primers. Two independently purified recombinant RSVB-GF virusisolates were extracted with an RNA extraction kit (Tel-Test,Friendswood, Tex.) and RNA was precipitated by isopropanol. Virion RNAswere annealed with a primer spanning the RSV region from nt 4468 to 4492and incubated for 1 hr under standard RT conditions (10 μl reactions)using superscript reverse transcriptase (Life Technologies,Gaithersburg, Md.). Aliquots of each reaction were subjected to PCR (30cycles at 94° C. for 30 s, 55° C. for 30 s and 72° C. for 2 min) usingsubgroup B specific primers in G region (CACCACCTACCTTACTCAAGT (SEQ IDNO:42) and TTTGTTTGTGGGTTTGATGGTTGG (SEQ ID NO:43)). The PCR productswere analyzed by electrophoresis on 1% agarose gel and visualized bystaining with ethidium bromide. As shown in FIG. 5, no DNA product wasproduced in RT/PCR reactions using RSV A2 strain as template. However, apredicted product of 254 bp was detected in RT/PCR reactions utilizingRSVB-GF RNA or the PCR control plasmid, pRSVB-GF DNA, as template,indicating the rescued virus contained G and F genes derived form B9320virus.

8.2 Expression of B9320G by RSV A2 Virus

RSV subgroup B strain B9320 G gene was amplified from B9320 vRNA byRT/PCR and cloned into pCRII vector for sequence determination. Two BglII sites were incorporated into the PCR primers which also containedgene start and gene end signals (GATATCAAGATCTACAATAACATTGGGGCAAATGC(SEQ ID NO:44) and GCTAAGAGATCTTTTTGAATAACTAAGCATG (SEQ ID NO:45)).B9320G cDNA insert was digested with Bgl II and cloned into the SH/G(4630 nt) or F/M2 (7552 nt) intergenic junction of a A2 cDNA subclone(FIG. 4B and FIG. 4C). The Xho I to Msc I fragment containing B9320Ginsertion either at SH/G or F/M2 intergenic region was used to replacethe corresponding Xho I to Msc I region of the A2 antigenomic cDNA. Theresulting RSV antigenomic cDNA clone was termed as pRSVB9320G-SH/G orpRSVB9320G-F/M2.

Generation of RSV A2 virus which had B9320 G gene inserted at F/M2intergenic region was performed similar to what has described forgeneration of RSVB-GF virus.

Briefly, pRSVB9320G-F/M2 together with plasmids encoding proteins N, Pand L were transfected, into Hep-2 cells, infected with a MVA vacciniavirus recombinant, which expresses the T7 RNA polymerase (LifeTechnologies, Gaithersburg, Md.). The transfected cell medium wasreplaced by MEM containing 2% fetal bovine serum (FBS) one day aftertransfection and further incubated for 3 days at 35° C. Aliquots ofculture supernatants (PO) were then used to infect fresh Hep-2 cells.After incubation for 6 days at 35° C., the supernatant was harvested andthe cells were fixed and stained by an indirect horseradish peroxidasemethod using goat anti-RSV antibody (Biogenesis) followed by a rabbitanti-goat antibody linked to horseradish peroxidase. The virus infectedcells were then detected by addition of substrate chromogen (Dako).RSV-like plaques were detected in the cells which were infected with thesupernatants from cells transfected with pRSVB9320G/F/M2.

Characterization of pRSVB9320G-F/M2 virus was performed by RT/PCR usingB9320G specific primers. A predicted PCR product of 410 bp was seen inRT/PCR sample using pRSVB9320G-F/M2 RNA as template, indicating therescued virus contained G gene derived from B9320. (FIG. 6)

Expression of the inserted RSV B9320 G gene was analyzed by Northernblot using a ³²P-labeled oligonucleotide specific to A2-G or B-G mRNA.Total cellular RNA was extracted from Hep-2 cells infected withwild-type RSVB 9320, rRSVA2, or rRSVB9320G-F/M2 48 hours postinfectionusing an RNA extraction kit (RNA stat-60, Tel-Test). RNA waselectrophoresed on a 1.2% agarose gel containing formaldehyde andtransferred to a nylon membrane (Amersham). An oligonucleotide specificto the G gene of the A2 stain(5′TCTTGACTGTTGTGGATTGCAGGGTTGACTTGACTCCGATCGATCC-3′ (SEQ ID NO:46)) andan oligonucleotide specific to the B9320 G gene(5′CTTGTGTTGTTGTTGTATGGTGTGTTTCTGATTTTGTATTGATCGATCC-3′ (SEQ ID NO:47))were labeled with ³²P-ATP by a kinasing reaction known to those ofordinary skill in the art. Hybridization of the membrane with one of the³²P-labeled G gene specific oligonucletodies was performed at 65° C. andwashed according to standard procedure. Both A2-G and B9320-G specificRNA were detected in the rRSVB9320G-F1M2 infected Hep-2 Cells. (FIG. 6B)These results demonstrate subtype specific RNA expression.

Protein expression of the chimeric rRSVA2(B-G) was compared to that ofRSV B9320 and rRSV by immunoprecipitation of ³⁵S-labeled infected Hep-2cell lysates. Briefly, the virus infected cells were labeled with³⁵S-promix (100 μCi/ml ³⁵S-Cys and ³⁵S-Met, Amersham, Arlington Heights,Ill.) at 14 hours to 18 hours post-infection according to a protocolknown to those of ordinary skill in the art. The cell monolayers werelysed by RIPA buffer and the polypeptides were immunoprecipitated witheither polyclonal antiserum raised in goat against detergent disruptedRSV A2 virus (FIG. 7, lanes 1-4) or antiserum raised in mice againstundisrupted B9320 virions (FIG. 7, lanes 5-8). The radio labeledimmunoprecipitated polypeptides were electrophoresed on 10%polyacrylamide gels containing 0.1% SDS and detected by autoradiography.Anti-RSV A2 serum immunoprecipitated the major polypeptides of the RSVA2 strain, whereas anti-B9320 serum mainly reacted with RSV B9320 Gprotein and the conserved F protein of both A and B subgroups. As shownin FIG. 7, a protein which is identical to the A2-G protein (lane 3),was immunoprecipitated from the rRSVA2(B-G) infected cells (lane 4) byusing an antiserum against RSV A2. The G protein of RSV B9320 strain wasnot recognized by the anti-A2 antiserum. A protein species, smaller thanA2-G protein, was immunoprecipitated from both B9320 (lane 6) andrRSVA2(B-G)(lane 9) infected cells using the antiserum raised in miceagainst B9320 virions. This polypeptide was not present in theuninfected and RSV A2 infected cells and likely is to represent the Gprotein specific to the RSV B 9320 strain. Amino acid sequencecomparison of both A2 and B9320 RSV G proteins indicated that twoadditional potential N-glycosylation sites (N—X—S/t) are present in theRSV A2G protein, which may contribute to slower migration of the A2 Gprotein under the conditions used. The F protein of RSV B9320 alsomigrated slightly faster than RSV A2 F protein. The P and M proteinsalso showed mobility differences between the two virus subtypes. Theidentity of the polypeptide near the top of the protein gel present inFSV B9320 and rRSVA2(B-G) infected cells is not known. Antisera raisedin mice against the RSV B9320 virions poorly recognized the N, P and Mproteins are compared to the goat antiserum raised against the RSV A2strain. The data described above clearly indicate that chimeric rRSVA2(B-G) expresses both the RSV A2 and B9320 specific G proteins.

8.2.1 Replication of Recombinant RSV in Tissue Culture

Recombinant RS viruses were plaque purified three times and amplified inHep-2 cells. Plaque assays were performed in Hep-2 cells in 12-wellplates using an overlay of 1% methylcellulose and 1×L15 mediumcontaining 2% fetal bovine serum (FBS). After incubation at 35° C. for 6days, the monolayers were fixed with methanol and plaques wereidentified by immunostaining. Plaque size and morphology of rRSV wasvery similar to that of wild-type A2 RSV (FIG. 8). However, the plaquesformed by rRSVC4G were smaller than rRSV and wild-type A2 virus. Theonly genetic difference between rRSV and rRSVC4 was a single nucleotidesubstitution in the RSV leader region. Therefore, the smaller plaquesize of rRSV A2(B-G) was not distinguishable from that of rRSVC4G.

The growth curves of rRSV, rRSVC4G and rRSV A2 (B-G) were compared tothat of the biologically derived wild-type A2 virus. Hep-2 cells weregrown in T25 culture flasks and infected with rRSV, rRSVC4G,rRSVA2(B-G), or wild-type RSV A2 strain at a moi of 0.5. After 1 houradsorption at 37° C., the cells were washed three times with MEMcontaining 2% FBS and incubated at 37° C. in 5% CO₂. At 4 hour intervalspost-infection, 250 μl of the culture supernatant was collected, andstored at −70° C. until virus titration. Each aliquot taken was replacedwith an equal amount of fresh medium. The titer of each virus wasdetermined by plaque assay on Hep-2 cells and visualized byimmunostaining (vide supra). As shown in FIG. 9, the growth kinetics ofrRSV is very similar to that of wild-type A2 virus. Maximum virus titerfor all the viruses were achieved between 48 hr to 72 hr. The virustiter of rRSVC4G was about 2.4-fold (at 48 hr) and 6.6-fold (at 72 hr)lower than rRSV and wild-type A2 RSV. The poor growth of rRSVC4G mayalso be due to the single nucleotide change in the leader region. Thechimeric rRSV A2(B-G) showed slower kinetics and lower peak titer (FIG.9).

9. EXAMPLE Generation of RSV L Gene Mutants

The strategy for generating L gene mutants is to introduce definedmutations or random mutations into the RSV L gene. The functionality ofthe L gene cDNA mutants can be screened in vitro by a minigenomereplication system. The recovered L gene mutants are then furtheranalyzed in vitro and in vivo.

9.1 Mutagenesis Strategies

9.1.1 Scanning Mutagenesis to Change the Clustered Charged Amino AcidsTo Alanine

This mutagenesis strategy has been shown to be particularly effective insystematically targeting functional domains exposed on protein surfaces.The rationale is that clusters of charged residues generally do not lieburied in the protein structure. Making conservative substitutions ofthese charged residues with alanines will therefore remove the chargeswithout grossly changing the structure of the protein. Disruption ofcharged clusters may interfere with the interaction of RSV L proteinwith other proteins and make its activity thermosensitive, therebyyielding temperature-sensitive mutants.

A cluster was originally defined arbitrarily as a stretch of 5 aminoacids in which two or more residues are charged residues. For scanningmutagenesis, all the charged residues in the clusters can be changed toalanines by site directed mutagenesis. Because of the large size of theRSV L gene, there are many clustered charged residues in the L protein.Therefore, only contiguous charged residues of 3 to 5 amino acidsthroughout the entire L gene were targeted (FIG. 10). The RSV L proteincontains 2 clusters of five contiguous charged residues, 2 clusters offour contiguous charged residues and 17 clusters of three contiguouscharge residues. Two to four of the charged residues in each clusterwere substituted with alanines.

The first step of the invention was to introduce the changes intopCITE-L which contains the entire RSV L-gene, using a QuikChangesite-directed mutagenesis kit (Stratagene). The introduced mutationswere then confirmed by sequence analysis.

9.1.2 Cysteine Scanning Mutagenesis

Cysteines are good targets for mutagenesis as they are frequentlyinvolved in intramolecular and intermolecular bond formations. Bychanging cysteines to glycines or alanines, the stability and functionof a protein may be altered because of disruption of its tertiarystructure. Thirty-nine cysteine residues are present in the RSV Lprotein (FIG. 11). Comparison of the RSV L protein with other members ofparamyxoviruses indicates that some of the cysteine residues areconserved.

Five conserved cysteine residues were changed to either valine(conservative change) or to aspartic acids (nonconservative change)using a QuikChange site-directed mutagenesis kit (Stratagene) degeneratemutagenic oligonucleotides. It will be apparent to one skilled in theart that the sequence of the mutagenic oligonucleotides is determined bythe protein sequence desired. The introduced mutations were confirmed bysequence analysis.

9.1.3 Random Mutagenesis

Random mutagenesis may change any residue, not simply charged residuesor cysteines. Because of the size of the RSV L gene, several L gene cDNAfragments were mutagenized by PCR mutagenesis. This was accomplished byPCR using exo Pfu polymerase obtained from Strategene. Mutagenized PCRfragments were then cloned into a pCITE-L vector. Sequencing analysis of20 mutagenized cDNA fragments indicated that 80%-90% mutation rates wereachieved. The functionality of these mutants was then screened by aminigenome replication system. Any mutants showing altered polymerasefunction were then further cloned into the full-length RSV cDNA cloneand virus recovered from transfected cells.

9.2 Functional Analysis of RSV L Protein Mutants by MinigenomeReplication System

The functionality of the L-genes mutants were tested by their ability toreplicate a RSV minigenome containing a CAT gene in its antisense andflanked by RSV leader and trailer sequences. Hep-2 cells were infectedwith MVA vaccinia recombinants expressing T7 RNA polymerase. After onehour, the cells were transfected with plasmids expressing mutated Lprotein together with plasmids expressing N protein and P protein, andpRSV/CAT plasmid containing CAT gene (minigenome). CAT gene expressionfrom the transfected cells was determined by a CAT ELISA assay(Boehringer Mannheim) according to the manufacturer's instruction. Theamount of CAT activity produced by the L gene mutant was then comparedto that of wild-type L protein.

9.3 Recovery of Mutant Recombinant RSV

To recover or rescue mutant recombinant RSV, mutations in the L-genewere engineered into plasmids encoding the entire RSV genome in thepositive sense (antigenome). The L gene cDNA restriction fragments (BamHI and Not I) containing mutations in the L-gene were removed from pCITEvector and cloned into the full-length RSV cDNA clone. The cDNA cloneswere sequenced to confirm that each contained the introduced mutations.

Each RSV L gene mutant virus was rescued by co-transfection of thefollowing plasmids into subconfluent Hep-2 cells grown in six-wellplates. Prior to transfection, the Hep-2 cells were infected with MVA, arecombinant vaccinia virus which expresses T7 RNA polymerase. One hourlater, cells were transfected with the following plasmids:

-   -   pCITE-N: encoding wild-type RSV N gene, 0.4 μg    -   pCITE-P: encoding wild-type RSV P gene, 0.4 μg    -   pCITE-Lmutant: encoding mutant RSV L gene, 0.2 μg    -   pRSVL mutant: full-length genomic RSV of the positive sense        (antigenome) containing the same L-gene mutations as pCITE-L        mutant, 0.6 μg

DNA was introduced into cells by lipofecTACE (Life Technologies) inOPTI-MEM. After five hours or overnight transfection, the transfectionmedium was removed and replaced with 2% MEM. Following incubation at 35°C. for three days, the media supernatants from the transfected cellswere used to infect Vero cells. The virus was recovered from theinfected Vero cells and the introduced mutations in the recoveredrecombinant viruses confirmed by sequencing of the RT/PCR DNA derivedfrom viral RNA.

Examples of the L gene mutants obtained by charged to alanine scanningmutagenesis are shown in the Table II. Mutants were assayed bydetermining the expression of CAT by pRSV/CAT minigenome followingco-transfection of plasmids expressing N, P and either wild-type ormutant L. Cells were harvested and lysed 40 hours post-transfectionafter incubation at 33° C. or 39° C. The CAT activity was monitored byCAT ELISA assay (Boehringer Mannheim). Each sample represents theaverage of duplicate transfections. The amount of CAT produced for eachsample was determined from a linear standard curve.

From the above preliminary studies, different types of mutations havebeen found.

9.3.1 Detrimental Mutations

Seven L protein mutants displayed a greater than 99% reduction in theamount of CAT produced compared to that of wild-type L protein. Thesemutations drastically reduced the activity of the RSV polymerase and arenot expected to be viable.

9.3.2 Intermediate Mutations

Several L mutants showed an intermediate level of CAT production whichranged from 1% to 50% of that wild-type L protein. A subset of thesemutants were introduced into virus and found to be viable. Preliminarydata indicated that mutant A2 showed 10-to 20-fold reduction in virustiter when grown at 40° C. compared 33° C. Mutant A25 exhibited asmaller plaque formation phenotype when grown at both 33° C. and 39° C.This mutant also had a 10-fold reduction in virus titer at 40° C.compared to 33° C.

9.3.3 Mutants with L Protein Function Similar or Higher than Wild Type LProtein

Some L gene mutants produced CAT gene expression levels similar to orgreater than the wild-type L protein in vitro and the recovered virusmutants have phenotypes indistinguishable from wild-type viruses intissue culture.

Once mutations in L that confer temperature sensitivity and attenuationhave been identified, the mutations will be combined to rest for thecumulative effect of multiple temperature-sensitivity markers. The Lmutants bearing more than one temperature sensitive marker are expectedto have lower permissive temperature and to be genetically more stablethan single-marker mutants.

The generated L gene mutants may also be combined with mutations presentin other RSV genes and/or with non-essential RSV gene deletion mutants(e.g., SH and M2-2 deletion). This will enable the selection of safe,stable and effective live attenuated RSV vaccine candidates.

10. GENERATION OF HUMAN RESPIRATORY SYNCYTIAL VIRUS VACCINE (RSV)CANDIDATE BY DELETING THE VIRAL SH AND M2 OR F2 GENES

10.1 M2-2 Deletion Mutant

To delete M2-2 genes, two Hind III restriction enzyme sites wereintroduced at RSV nucleotides 8196 and 8430, respectively, in a cDNAsubclone pET(S/B) which contained an RSV restriction fragment from 4478to 8505. The RSV restriction fragment had been previously prepared byQuikchange site-directed mutagenesis (Strategene, Lo Jolla, Calif.).Digestion of pET(S/B) with Hind III restriction enzyme removed a 234nucleotide sequence which contained the majority of the M2-2 openreading frame. The nucleotides encoding the first 13 amino acids at theN-terminus of the M2-2 gene product were not removed because thissequence overlaps M2-1. The cDNA fragment which contained M2-2 genedeletion was digested with SacI and BamHI and cloned back into afull-length RSV cDNA clone, designated pRSVΔM2-2

Infectious RSV with this M2-2 deletion was generated by transfectingpRSVΔM2-2 plasmid into MVA-infected Hep-2 cells expressing N, P and Lgenes. Briefly, pRSVΔM2-2 was transfected, together with plasmidsencoding proteins N, P and L, into Hep-2 cells which had been infectedwith a recombinant vaccinia virus (MVA) expressing the T7 RNApolymerase. Transfection and recovery of recombinant RSV was performedas follows. Hep-2 cells were split five hours or a day before thetransfection in six-well dishes. Monolayers of Hep-2 cells at 70% -80%confluence were infected with MVA at a multiplicity of infection (moi)of 5 and incubated at 35° C. for 60 min. The cells were then washed oncewith OPTI-MEM (Life Technologies, Gaithersburg, Md.). Each dish wasreplaced with 1 ml of OPTI-MEM and 0.2 ml transfection medium was added.The transfection medium was prepared by mixing 0.5-0.6 μg of RSVantigenome, 0.4 μg of N plasmid, 0.4 μg of P plasmid, and 0.2 μg of Lplasmid in a final volume of 0.1 ml OPTI-MEM medium. This was combinedwith 0.1 ml of OPTI-MEM containing 10 μl lipofecTACE (LifeTechnologies). After a 15 minute incubation at room temperature, theDNA/lipofecTACE mixture was added to the cells. The medium was replacedone day later with MEM containing 2% FBS. Cultures were furtherincubated at 35° C. for 3 days and the supernatants harvested. Threedays post-transfection, 0.3-0.4 ml culture supernatants were passagedonto fresh Hep-2 cells and incubated with MEM containing 2% FBS. Afterincubation for six days, the supernatant was harvested and the cellswere fixed and stained by an indirect horseradish peroxidase methodusing goat anti-RSV antibody (Biogenesis) followed by a rabbit anti-goatantibody linked to horseradish peroxidase. The virus infected cells werethen detected by addition of substrate chromogen (DAKO) according to themanufacturer's instructions. Recombinant RSV which contained M2-2 genedeletion was recovered from the transfected cells. Identification ofrRSVΔM2-2 was performed by RT/PCR using primers flanking the deletedregion. As shown in FIG. 12A, a cDNA fragment which is 234 nucleotidesshorter than the wild-type RSV was detected in rRSVΔM2-2 infected cells.No cDNA was detected in the RT/PCR reaction which did not containreverse transcriptase in the RT reaction. This indicated that the DNAproduct was derived from viral RNA and not from contamination. Theproperties of the M2-2 deletion RSV are currently being evaluated.

10.2 SH Deletion Mutant.

To delete the SH gene from RSV, a Sac I restriction enzyme site wasintroduced at the gene start signal of SH gene at position of nt 4220. Aunique SacI site also exists at the C-terminus of the SH gene.Site-directed mutagenesis was performed in subclone pET(A/S), whichcontains an AvrII(2129) SacI (4478) restriction fragment. Digestion ofpET(A/S) mutant with SacI removed a 258 nucleotide fragment of the SHgene. Digestion of the pET(A/S) subclone containing the SH deletion wasdigested with Avr II and Sac I and the resulting restriction fragmentwas then cloned into a full-length RSV cDNA clone. The full-length cDNAclone containing the SH deletion was designated pRSVΔSH.

Generation of the pRSVΔSH mutant was performed as described above (see10.1). SH-minus RSV (rRSVΔASH) was recovered from MVA-infected cellsthat had been co-transfected with pRSVΔSH together with N, P and Lexpression plasmids. Identification of the recovered rRSVΔSH wasperformed by RT/PCR using a pair of primers which flanked the SH gene.As shown in FIG. 12A, a cDNA band which is about 258 nucleotides shorterthan the wild-type virus was detected in the rRSVΔSH infected cells. NoDNA was detected in the RT/PCR reaction which did not have reversetranscriptase in the RT reaction. This indicated that the PCR DNA wasderived from viral RNA and was not artifact, and that the virus obtainedwas truly SH-minus RSV.

10.3 Generation of Both SH and M2-2 Deletion Mutant.

Both SH and M2-2 genes were deleted from the full-length RSV cDNAconstruct by cDNA subcloning. A Sac I to Bam HI fragment containing M2-2deletion removed from cDNA subclone pET(S/B)ΔM2-2RSV was cloned intopRSVΔSH cDNA clone. The double gene deletion plasmid pRSVΔSHΔM2-2 wasconfirmed by restriction enzyme mapping. As shown in FIG. 12B, theSH/M2-2 double deletion mutant is shorter than the wild-type pRSV cDNA.

Recovery of infectious RSV containing both the SH and M2-2 deletion wasperformed as described earlier. Infectious virus with both SH and M2-2deleted was obtained from transfected Hep-2 cells. TABLE II CATExpression levels of Mutant RSV L-gene Conc. of CAT (ng/mL) ChargeCharge to Alanine Rescued Mut. 33° C. 39° C. Cluster Change Virus A330.246 Bkg 5 135E, 136K No A73 3.700 0.318 3 146D, 147E, 148 D Yes A1713.020 Bkg 3 157K, 158D Yes A81 1.000 0.280 3 255H, 256K Yes A185 Bkg Bkg3 348E, 349E No A91 Bkg Bkg 3 353R, 355R No A101 Bkg Bkg 3 435D, 436E,437R No A192 1.960 Bkg 3 510E, 511R Yes A11 0.452 Bkg 1 520R Yes A1112.320 0.267 4 568H, 569E Yes A121 0.772 Bkg 2 587L, 588R No A133 Bkg Bkg4 620E, 621R No A141 2.800 Bkg 3 1025K, 1026D Yes A25 0.169 Bkg 3 1033D,1034D Yes A45 5.640 0.478 5 1187D, 1188K Yes A153 4.080 0.254 5 1187D,1188K, 1189R, Yes 1190E A162 10.680  Bkg 3 1208E, 1209R No A201 Bkg Bkg3 1269E, 1270K No A211 2.440 0.047 3 1306D, 1307E Yes A221 0.321 Bkg 31378D, 1379E No A231 Bkg Bkg 3 1515E, 1516K No A241 1.800 0.308 3 1662H,1663K Yes A57 5.660 0.706 3 1725D, 1726K Yes A65 3.560 0.168 2 1957R,1958K Yes A251 0.030 Bkg. 3 2043D, 2044K Yes A261 Bkg Bkg 3 2102K, 2103HNo AD11 2.800 0.456 5 and 3 1187D, 1188K, 1725D, No 1726K AD21 2.6400.226 5 and 2 1187D, 1188K, 1957R, No 1958K AD31 1.280 0.192 3 and 21725D, 1726K, 1957R, No 1958K F1 Bkg Bkg — 521 F to L Yes F13 0.13  Bkg— 521 F to L Yes Lwt 3.16  — — no amino acid changes Yes

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any constructs, viruses orenzymes which are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method for rescuing a genetically manipulated non-segmentednegative-stranded RNA virus of the paramyxoviridae family comprising:introducing into a host cell expressing a heterologous RNA polymerase:(a) one or more DNA molecules encoding the virus N, P and L proteinsoperably linked to a polymerase binding site; (b) a DNA moleculecomprising the cDNA of the non-segmented negative stranded RNA viruswherein the cDNA encodes the entire genome of the virus or is modifiedby the incorporation of a mutation or a heterologous sequence, andwherein the cDNA is transcribed by a heterologous RNA polymerase; andisolating the virus produced by the cell.
 2. The method of claim 1,wherein the virus is a parainfluenza virus.
 3. The method of claim 1,wherein the virus is a respiratory syncytial virus.